Use of aerosolized levofloxacin for treating cystic fibrosis

ABSTRACT

Methods for treating cystic fibrosis. The method includes administering to a human in need thereof an aerosol solution comprising levofloxacin or ofloxacin and a divalent or trivalent cation. More particularly, the method includes administering the aerosol solution to a human having a pulmonary infection comprising P. aeruginosa.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/412,423 filed Mar. 5, 2012, allowed, which is a continuation of PCTInternational Application No. PCT/US2010/047903 filed Sep. 3, 2010,which claims priority to U.S. Application No. 61/240,092 filed Sep. 4,2009, and U.S. Application No. 61/249,231 filed Oct. 6, 2009, which arehereby expressly incorporated by reference in their entireties.

FIELD OF THE INVENTION

Methods and compositions for treating cystic fibrosis are provided. Inparticular, compositions and methods for the use of aerosolizedlevofloxacin for treating cystic fibrosis are provided.

BACKGROUND

Patients with cystic fibrosis (CF) suffer from chronic infections of thelower respiratory tract that can be caused by bacteria, includingPseudomonas aeruginosa. Pseudomonas aeruginosa has been particularlyproblematic to eradicate and has been implicated as the major cause ofmorbidity and mortality in CF patients.

Aerosol delivery of antibiotics directly to the lungs has the potentialto increase the local concentration of antibiotic at the site ofinfection, thereby, enhancing bacterial killing compared with systemicadministration. Currently, tobramycin solution for inhalation is theonly aerosol antibiotic approved for the management of CF patients withbacteria such as P aeruginosa. Because of the development of resistanceto tobramycin and the limited effect on reducing bacterial density insputum, there is a need for improved therapies to treat CF patients withpulmonary infections caused by multidrug resistant bacteria, including Paeruginosa.

SUMMARY

Some embodiments of the present invention relate to compositions, use ofsuch compositions, and methods for treating cystic fibrosis. Some suchembodiments include compositions and methods for the use of aerosolizedlevofloxacin for treating cystic fibrosis.

Some embodiments include methods for treating cystic fibrosis in a humanin which the human has a pulmonary infection comprising P. aeruginosa.In some such embodiments, the methods includes administering to saidhuman in need thereof an aerosol of a solution comprising levofloxacinor ofloxacin and a divalent or trivalent cation to achieve a reductionin the density of the P. aeruginosa in the sputum of said human by atleast 40%, at least 44%, at least 70%, at least 900/% and at least 97%.Some such embodiments include achieving a reduction in the density ofthe P. aeruginosa in the sputum of said human by at least 0.25 log₁₀CFU/g sputum, at least 0.50 log₁₀ CFU/g sputum, at least 1.0 log₁₀ CFU/gsputum, at least 1.5 log₁₀ CFU/g sputum, and at least 1.8 log₁₀ CFU/gsputum.

Some embodiments include methods for treating cystic fibrosis in a humanthat include administering to the human in need thereof an aerosol of asolution comprising levofloxacin or ofloxacin and a divalent ortrivalent cation to achieve an increase in FEV₁ of at least 2% and anincrease in FEF 25-75 of at least 5%, an increase in FEV₁ of at least 5%and an increase in FEF 25-75 of at least 10%, an increase in FEV₁ of atleast 7% and an increase in FEF 25-75 of at least 15%, and an increasein FEV₁ of at least 10%, and an increase in FEF 25-75 of at least 20%.

Some embodiments include achieving an increase in FEV₁ of at least 0.05L and an increase in FEF 25-75 of at least 0.05 L, an increase in FEV₁of at least 0.10 L and an increase in FEF 25-75 of at least 0.10 L, anincrease in FEV₁ of at least 0.15 L and an increase in FEF 25-75 of atleast 0.15 L, an increase in FEV₁ of at least 0.20 L and an increase inFEF 25-75 of at least 0.20 L, and an increase in FEV₁ of at least 0.25 Land an increase in FEF 25-75 of at least 0.25 L. Some embodimentsinclude achieving an increase in FEF 25-75 of at least 0.27 L.

Some embodiments include methods for treating cystic fibrosis in a humanthat include administering to said human in need thereof an aerosol of asolution comprising levofloxacin or ofloxacin and a divalent ortrivalent cation to achieve a hazard ratio less than 1.0, wherein thehazard ratio is indicative of a decreased need for otheranti-pseudomonal antimicrobials. In some such embodiments, the hazardratio is less than 0.8, less than 0.6, less than 0.4, and less than 0.3.

Some embodiments include methods of treating cystic fibrosis in a humanwho is being administered an agent by inhalation selected from the groupconsisting of one or more of dornase alpha, azithromycin, salbutamol,pancrelipase, sodium chloride, seretide, and ADEK, comprisingadministering to said human an aerosol of a solution comprisinglevofloxacin or ofloxacin and a divalent or trivalent cation. In someembodiments, the agent is selected from the group consisting ofsalbutamol, pancrelipase, seretide, and ADEK. In some embodiments, thehuman has a pulmonary infection comprising P. aeruginosa. Someembodiments include achieving a reduction in the density of the P.aeruginosa in the sputum of said human by at least 0.25 log₁₀ CFU/gsputum, at least 0.50 log₁₀ CFU/g sputum, and at least 1.0 log₁₀ CFU/gsputum.

Some embodiments include methods for treating cystic fibrosis in a humanin which the human has a pulmonary infection comprising P. aeruginosa.Some such embodiments include repeatedly administering to said human inneed thereof an aerosol of a solution comprising levofloxacin orofloxacin and a divalent or trivalent cation, wherein said repeatedadministration does not result in a greater than 16-fold increase inminimum inhibitory concentration (MIC) of the P. aeruginosa strain insaid human having the highest MIC relative to other P. aeruginosastrains. In some embodiments, the repeated administration does notresult in a greater than 8-fold increase in minimum inhibitoryconcentration (MIC) of the P. aeruginosa strain in said human having thehighest MIC relative to other P. aeruginosa strains. In someembodiments, repeated administration does not result in a greater than4-fold increase in minimum inhibitory concentration (MIC) of the P.aeruginosa strain in said human having the highest MIC relative to otherP. aeruginosa strains.

Some embodiments include methods for treating cystic fibrosis in a humanin which the human has a pulmonary infection comprising P. aeruginosa.Some such embodiments include repeatedly administering to said human inneed thereof an aerosol of a solution comprising levofloxacin orofloxacin and a divalent or trivalent cation to achieve an increase in aCFQ-R respiratory domain greater than 1, greater than 2, greater than 3,greater than 4, and greater than 5.

Some embodiments include methods of decreasing small airway resistancein a human with cystic fibrosis, comprising administering to said humanin need thereof an aerosol of a solution comprising levofloxacin orofloxacin and a divalent or trivalent cation to achieve an increase inFEF 25-75 of at least 5%, at least 10%, at least 15%, and at least 20%.Some embodiments include, achieving an increase in FEF 25-75 of at least0.05 L, at least 0.10 L, at least 0.15 L, at least 0.20 L, at least 0.25L, and at least 0.27 L.

Some embodiments include methods of administering levofloxacin orofloxacin to a human, comprising repeatedly administering to said humanan aerosol of a solution comprising levofloxacin or ofloxacin and adivalent or trivalent cation, wherein said repeated administration doesnot result in an incidence of arthralgia. In some methods, administeringis repeated at least once daily for 14 days, at least once daily for 28days, and at least once daily for 35 days. In some such methods,administering is repeated at least twice daily for 14 days, at leasttwice daily for 28 days, and at least twice daily for 35 days.

Some embodiments include methods for treating cystic fibrosis in a humanin which the human has a pulmonary infection comprising P. aeruginosaand said human has a body surface area less than 1.5 m². Some suchmethods include administering to said human in need thereof an aerosolof a solution comprising levofloxacin or ofloxacin and a divalent ortrivalent cation to achieve a dose-normalized serum AUC at least 20(ng·h/L)/mg Dose. In some embodiments, the administering is repeateddaily for at least 14 days the aerosol comprises an amount oflevofloxacin or ofloxacin of at least 80 mg, 120 mg, 180 mg, and 240 mg.Some embodiments include achieving a dose-normalized serum AUC at least20 (ng·h/L)/mg Dose, at least 40 (ng·h/L)/mg Dose, at least 60(ng·h/L)/mg Dose, at least 80 (ng·h/L)/mg Dose, and at least 100(ng·h/L)/mg Dose.

Some embodiments include methods for treating cystic fibrosis in a humanin which the human has a pulmonary infection comprising P. aeruginosaand the human has a body surface area less than 1.5 m₂. Some suchmethods include administering to said human in need thereof an aerosolof a solution comprising levofloxacin or ofloxacin and a divalent ortrivalent cation to achieve a dose-normalized serum C_(max) greater than2 μg/L/mg administered dose greater than 4 μg/L/mg administered dose,greater than 6 μg/L/mg administered dose, greater than 8 μg/L/mgadministered dose, and greater than 14 μ/L/mg administered dose. In someembodiments, the human is less than 15 years of age, less than 12 yearsof age, less than 10 years of age. In some embodiments, the humancomprises a mass less than 55 kg, less than 45 kg, less than 35 kg, lessthan 25 kg.

In some of the foregoing embodiments, the solution consists essentiallyof levofloxacin or ofloxacin and the divalent or trivalent cation.

In some of the foregoing embodiments, the solution comprises no lactose.

In some of the foregoing embodiments, the solution comprises a divalentor trivalent cation concentration from about 50 mM to about 400 mM, anda levofloxacin or ofloxacin concentration from between about 50 mg/ml toabout 200 mg/ml.

In some of the foregoing embodiments, the solution comprises a divalentor trivalent cation concentration from about 100 mM to about 300 mM, anda levofloxacin or ofloxacin concentration from between about 75 mg/ml toabout 150 mg/ml.

In some of the foregoing embodiments, the solution comprises a divalentor trivalent cation concentration from about 150 mM to about 250 mM, anda levofloxacin or ofloxacin concentration from between about 90 mg/ml toabout 125 mg/ml.

In some of the foregoing embodiments, wherein the solution comprises anosmolality from about 300 mOsmol/kg to about 500 mOsmol/kg, and a pHfrom about 5 to about 8.

In some of the foregoing embodiments, the solution comprises anosmolality from about 350 mOsmol/kg to about 425 mOsmol/kg, and a pHfrom about 5 to about 6.5.

In some of the foregoing embodiments, the solution comprises a pH fromabout 5.5 to about 6.5.

In some of the foregoing embodiments, the divalent or trivalent cationis selected from magnesium, calcium, zinc, copper, aluminum, and iron.

In some of the foregoing embodiments, the solution comprises magnesiumchloride.

In some of the foregoing embodiments, the solution comprises alevofloxacin or ofloxacin concentration between about 90 mg/ml to about110 mg/ml, a magnesium chloride concentration between about 175 mM toabout 225 mM, a pH between about 5 to about 7; an osmolarity of betweenabout 300 mOsmol/kg to about 500 mOsmol/kg, and lacks lactose.

In some of the foregoing embodiments, the aerosol comprises a massmedian aerodynamic diameter from about 2 microns to about 5 microns witha geometric standard deviation less than or equal to about 2.5 microns.

In some of the foregoing embodiments, the aerosol comprises a massmedian aerodynamic diameter from about 2.5 microns to about 4.5 micronswith a geometric standard deviation less than or equal to about 1.8microns.

In some of the foregoing embodiments, the aerosol comprises a massmedian aerodynamic diameter from about 2.8 microns to about 4.3 micronswith a geometric standard deviation less than or equal to about 2microns.

Some of the foregoing embodiments also include producing the aerosolwith a vibrating mesh nebulizer. In some such embodiments, the vibratingmesh nebulizer is a PARI E-FLOW® nebulizer.

In some of the foregoing embodiments, the amount of levofloxacin orofloxacin administered to the lung is at least about 20 mg, at leastabout 100 mg, at least about 125 mg, and at least about 150 mg

In some of the foregoing embodiments, at least about 100 mg the aerosolis administered to the lung in less than about 10 minutes, less thanabout 5 minutes, less than about 3 minutes, less than about 2 minutes.

Some of the foregoing embodiments also include co-administering anadditional active agent selected from the group consisting ofantibiotics, bronchodilators, anticholinergics, glucocorticoids,eicosanoid inhibitors, and combinations thereof

In some embodiments, the antibiotic can include tobramycin, aztreonam,ciprofloxacin, azithromycin, tetracycline, quinupristin, linezolid,vancomycin, and chloramphenicol, colisitin and combinations thereof. Insome embodiments, the bronchodilator can include salbutamol,levosalbuterol, terbutaline, fenoterol, terbutlaine, pirbuterol,procaterol, bitolterol, rimiterol, carbuterol, tulobuterol, reproterol,salmeterol, formoterol, arformoterol, bambuterol, clenbuterol,indacterol, theophylline, roflumilast, cilomilast, and combinationsthereof. In some embodiments, the anticholinergic can includeipratropium, tiotropium, and combinations thereof. In some embodiments,the glucocorticoid can include prednisone, fluticasone, budesonide,mometasone, ciclesonide, beclomethasone, and combinations thereof insome embodiments, the eicosanoid can include montelukast, pranlukast,zafirlukast, zileuton, ramatroban, seratrodast, and combinationsthereof. In some embodiments, co-administering comprises inhaling theadditional active agent.

Some of the foregoing embodiments include administering the aerosol oncedaily.

Some of the foregoing embodiments administering the aerosol twice daily.

In some of the foregoing embodiments, the pulmonary infection furthercomprises one or more bacteria that can include Pseudomonas fluorescens,Pseudomonas acidovorans, Pseudomonas alcaligenes, and Pseudomonasputida, Stenotrophomonas maltophilia, Aeromonas hydrophilia, Escherichiacoli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi,Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae,Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacteraerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratiamarcescens, Morganella morganii, Proteus mirabilis, Proteus vulgaris,Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii,Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersiniaenterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersiniaintermedia, Bordetella pertussis, Bordetella parapertussis, Bordetellabronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae,Haemophilus haemolyticus, Haemophilus parahaemolvticus, Haemophilusducreyi. Pasteurella multocida, Pasteurella haemolytica, Helicobacterpylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli,Borrelia burgdorferi, Vibrio cholera, Vibrio parahaemolyticus,Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae,Neisseria meningitidis, Burkholderia cepacia, Francisella tularensis,Kingella, and Morarella.

In some of the foregoing embodiments, the pulmonary infection furthercomprises a gram-negative anaerobic bacteria.

In some of the foregoing embodiments, the pulmonary infection furthercomprises one or more of the bacteria that can include Bacteroidesfragilis, Bacteroides distasonis, Bacteroides 3452A homology group,Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron,Bacteroides uniformis, Bacteroides eggerthii, and Bacteroidessplanchnicus.

In some of the foregoing embodiments, the pulmonary infection furthercomprises a gram-positive bacteria.

In some of the foregoing embodiments, the pulmonary infection furthercomprises one or more of the bacteria that can include Corynebacteriumdiphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae,Streptococcus agalactiae. Streptococcus pyogenes, Streptococcus milleri;Streptococcus (Group G); Streptococcus (Group C/F); Enterococcusfaecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcusepidermidis, Staphylococcus saprophyticus. Staphylococcus intermedius,Staphylococcus hyicus subsP. hyicus, Staphylococcus haemolyticus,Staphylococcus hominis, and Staphylococcus saccharolyticus.

In some of the foregoing embodiments, the pulmonary infection furthercomprises a gram-positive anaerobic bacteria.

In some of the foregoing embodiments, the pulmonary infection furthercomprises one or more bacteria that can include Clostridium difficile,Clostridium perfringens. Clostridium tetini, and Clostridium botulinum.

In some of the foregoing embodiments, the pulmonary infection furthercomprises an acid-fast bacteria.

In some of the foregoing embodiments, the pulmonary infection furthercomprises one or more bacteria that can include Mycobacteriumtuberculosis, Mycobacterium avium, Mycobacterium intracellulare, andMycobacterium leprae.

In some of the foregoing embodiments, the pulmonary infection furthercomprises an atypical bacteria.

In some of the foregoing embodiments, the pulmonary infection furthercomprises one or more bacteria that can include Chlamydia pneumoniae andMycoplasma pneumoniae.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show graphs using linear axes and semi-logarithmic axes,respectively, of the arithmetic mean serum concentrations oflevofloxacin after administration of single 180 mg doses as a 50 mg/mland 100 mg/ml solution for inhalation and after administration of 240 mgas a 100 mg/ml solution for inhalation once daily for 7 days to patientswith CF.

FIGS. 2A and 2B show graphs using linear axes and semi-logarithmic axes,respectively, of the arithmetic mean sputum concentrations oflevofloxacin after administration of single 180 mg doses as a 50 mg/mland 100 mg/ml solution for inhalation and after administration of 240 mgas a 100 mg/ml solution for inhalation once daily for 7 days to patientswith CF.

FIG. 3 shows a graph of the mean change in P. aeruginosa (log 10 CFU/gsputum) over time for various treatment arms in an EE patientpopulation. Patients were administered: levofloxacin 120 mg QD,levofloxacin 240 mg QD, levofloxacin 240 mg BID, or placebo.

FIG. 4 shows a graph of change in P. aeruginosa (log 10 CFU/g sputum)over time for various treatment arms in a MITT patient population.Patients were administered: levofloxacin 120 mg QD, levofloxacin 240 mgQD, levofloxacin 240 mg BID, or placebo.

FIG. 5 shows a graph of survival distribution function over time forvarious treatment groups in an EE population.

FIG. 6 shows a graph of survival distribution function over time forvarious treatment groups in a MITT population.

FIG. 7 shows a graph of percent change in FEV₁(L) at Day 28 for the EEpopulation treated with placebo, 120 mg QD, 240 mg QD, or 240 mg BID.

FIG. 8 shows a graph of percent change in FEV₁(L) vs. placebo at Day 28for the EE population treated with 120 mg QD, 240 mg QD, or 240 mg BID.*p=0.0102.

FIG. 9 shows a graph of the categorical change in percent predicted FEV₁at Day 28 for the EE population treated with 120 mg QD, 240 mg QD, or240 mg BID. *p=0.0370, **p=0.0037.

FIG. 10 shows a graph of dose normalized serum AUC (ng·h/L/mgDose) vs.pediatric CF patient body weight (▪ 180 mg (n=7); ♦ 240 mg (n=18)). Themean value is shown for serum levofloxacin AUC in adult CF patientsstudied in another clinical trial (not shown) and based on a mean weightof 71 kg (▴; +/−standard deviation).

FIG. 11A shows a graph of dose normalized serum AUC (ng·h/L/mgDose) vs.pediatric CF patient age (▪ 180 mg; ♦ 240 mg). FIG. 11B shows a graph ofdose normalized serum AUC (ng·h/L/mg Dose) vs. BSA (body surface area)(▪ 180 mg;” ♦ 240 mg). FIG. 11C shows a graph of dose normalized serumC_(max) (μ/L/mg administered dose) vs. pediatric CF patient body weight(▪ 180 mg; ♦ 240 mg). FIG. 11D shows a graph of dose normalized serumC_(max) (μ/L/mg administered dose) vs. pediatric CF patient age (▪ 180mg; ♦ 240 mg). FIG. 11E shows a graph of dose normalized serum C_(max)(μg/L/mg administered dose) vs. BSA (▪ 180 mg; ♦ 240 mg).

DETAILED DESCRIPTION

The present invention relates to the use of particular formulations oflevofloxacin solutions for inhalation and particular dosage andadministration regimens for the treatment of cystic fibrosis. In someembodiments, treatment of cystic fibrosis includes treatment ofpulmonary infections associated with cystic fibrosis, such as P.aeruginosa, S. pneumoniae, H influenzae, Burkholderia sp.,Staphylococcus aureus, Stenotrophomonas sp., and M catarrhalis. Methodsdescribed herein for treating cystic fibrosis can include administeringformulations of levofloxacin. In some embodiments, methods for treatingcystic fibrosis can also include achieving a reduction in the density ofparticular pathogens in the lungs of a subject. In some embodiments,methods for treating cystic fibrosis can also include improvingpulmonary characteristics of a subject that can be measured withparameters such as FEV₁, FEF 25-72, and the like.

The present invention provides several advantages. For example, aerosollevofloxacin provides high doses of drug directly to the lung. Highdoses are advantageous in reducing the development of resistant strains.In addition, the present invention provides formulations with reducedadverse effects typically associated with fluoroquinolones, such asarthralgia. Some embodiments provide methods for treating cysticfibrosis that decrease the risk of acute exacerbations in CF patients atrisk for exacerbations. More embodiments provide methods for increasingthe airflow in the lungs of CF patients. In various embodiments, theabove methods are achieved by administering aerosolized levofloxacin orofloxacin in dosages and administration schedules sufficient to achievethe recited result.

Definitions

The term “administration” or “administering” refers to a method ofgiving a dosage of an antimicrobial pharmaceutical composition to avertebrate. The preferred method of administration can vary depending onvarious factors, e.g., the components of the pharmaceutical composition,the site of the potential or actual bacterial infection, the microbeinvolved, and the severity of an actual microbial infection. In someembodiments, administration can include loading an instrument to delivera drug to a subject. In some such embodiments, administering can includeloading a nebulizer with a drug to be delivered to a patient. Thus, thedosage administered may be the dosage loaded into the instrument (e.g.,nebulizer).

A “carrier” or “excipient” is a compound or material used to facilitateadministration of the compound, for example, to increase the solubilityof the compound. Solid carriers include, e.g., starch, lactose,dicalcium phosphate, sucrose, and kaolin. Liquid carriers include, e.g.,sterile water, saline, buffers, non-ionic surfactants, and edible oilssuch as oil, peanut and sesame oils. In addition, various adjuvants suchas are commonly used in the art may be included. These and other suchcompounds are described in the literature, e.g., in the Merck Index,Merck & Company, Rahway, N.J. Considerations for the inclusion ofvarious components in pharmaceutical compositions are described, e.g.,in Gilman et al. (Eds.) (1990); Goodman and Gilman's: ThePharmacological Basis of Therapeutics, 8th Ed., Pergamon Press,incorporated by reference herein in its entirety.

A “diagnostic” as used herein is a compound, method, system, or devicethat assists in the identification and characterization of a health ordisease state. The diagnostic can be used in standard assays as is knownin the art.

The term “mammal” is used in its usual biological sense. Thus, itspecifically includes humans, cattle, horses, dogs, and cats, but alsoincludes many other species.

The term “microbial infection” refers to the undesired proliferation orpresence of invasion of pathogenic microbes in a host organism. Thisincludes the excessive growth of microbes that are normally present inor on the body of a mammal or other organism. More generally, amicrobial infection can be any situation in which the presence of amicrobial population(s) is damaging to a host mammal. Thus, a microbialinfection exists when excessive numbers of a microbial population arepresent in or on a mammal's body, or when the effects of the presence ofa microbial population(s) is damaging the cells or other tissue of amammal.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The term “pharmaceutically acceptable salt” refers to salts that retainthe biological effectiveness and properties of the compounds of thisinvention and, which are not biologically or otherwise undesirable. Inmany cases, the compounds of this invention are capable of forming acidand/or base salts by virtue of the presence of amino and/or carboxylgroups or groups similar thereto. Pharmaceutically acceptable acidaddition salts can be formed with inorganic acids and organic acids.Inorganic acids from which salts can be derived include, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Organic acids from which salts can bederived include, for example, acetic acid, propionic acid, naphtoicacid, oleic acid, palmitic acid, pamoic (emboic) acid, stearic acid,glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, ascorbic acid,glucoheptonic acid, glucuronic acid, lactic acid, lactobioic acid,tartaric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like. Pharmaceutically acceptable base additionsalts can be formed with inorganic and organic bases. Inorganic basesfrom which salts can be derived include, for example, sodium, potassium,lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese,aluminum, and the like; particularly preferred are the ammonium,potassium, sodium, calcium and magnesium salts. Organic bases from whichsalts can be derived include, for example, primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines, basic ion exchange resins, and thelike, specifically such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, histidine, arginine, lysine, benethamine,N-methyl-glucamine, and ethanolamine. Other acids include dodecylsufuricacid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, andsaccharin.

“Solvate” refers to the compound formed by the interaction of a solventand fluoroquinolone antimicrobial, a metabolite, or salt thereof.Suitable solvates are pharmaceutically acceptable solvates includinghydrates.

In the context of the response of a microbe, such as a bacterium, to anantimicrobial agent, the term “susceptibility” refers to the sensitivityof the microbe for the presence of the antimicrobial agent. So, toincrease the susceptibility means that the microbe will be inhibited bya lower concentration of the antimicrobial agent in the mediumsurrounding the microbial cells. This is equivalent to saying that themicrobe is more sensitive to the antimicrobial agent. In most cases theminimum inhibitory concentration (MIC) of that antimicrobial agent willhave been reduced. The MIC₉₀ can include the concentration to inhibitgrowth in 90% of organisms.

By “therapeutically effective amount” or “pharmaceutically effectiveamount” is meant a fluoroquinolone antimicrobial agent, as disclosed forthis invention, which has a therapeutic effect. The doses offluoroquinolone antimicrobial agent which are useful in treatment aretherapeutically effective amounts. Thus, as used herein, atherapeutically effective amount means those amounts of fluoroquinoloneantimicrobial agent which produce the desired therapeutic effect asjudged by clinical trial results and/or model animal infection studies.In particular embodiments, the fluoroquinolone antimicrobial agent areadministered in a pre-determined dose, and thus a therapeuticallyeffective amount would be an amount of the dose administered. Thisamount and the amount of the fluoroquinolone antimicrobial agent can beroutinely determined by one of skill in the art, and will vary,depending on several factors, such as the particular microbial straininvolved. This amount can further depend upon the patient's height,weight, sex, age and medical history. For prophylactic treatments, atherapeutically effective amount is that amount which would be effectiveto prevent a microbial infection.

A “therapeutic effect” relieves, to some extent, one or more of thesymptoms of the infection, and includes curing an infection. “Curing”means that the symptoms of active infection are eliminated, includingthe total or substantial elimination of excessive members of viablemicrobe of those involved in the infection to a point at or below thethreshold of detection by traditional measurements. However, certainlong-term or permanent effects of the infection may exist even after acure is obtained (such as extensive tissue damage). As used herein, a“therapeutic effect” is defined as a statistically significant reductionin bacterial load in a host, emergence of resistance, or improvement ininfection symptoms as measured by human clinical results or animalstudies.

“Treat,” “treatment,” or “treating,” as used herein refers toadministering a pharmaceutical composition for prophylactic and/ortherapeutic purposes. The term “prophylactic treatment” refers totreating a patient who is not yet infected, but who is susceptible to,or otherwise at risk of, a particular infection such that there is areduced onset of infection. The term “therapeutic treatment” refers toadministering treatment to a patient already suffering from aninfection. Thus, in preferred embodiments, treating is theadministration to a mammal (either for therapeutic or prophylacticpurposes) of therapeutically effective amounts of a fluoroquinoloneantimicrobial agent.

Pharmacokinetics (PK) is concerned with the time course of antimicrobialconcentration in the body. Pharmacodynamics (PD) is concerned with therelationship between pharmacokinetics and the antimicrobial efficacy invivo. PK/PD parameters correlate antimicrobial exposure withantimicrobial activity. The rate of killing by antimicrobial isdependent on antimicrobial mode of action and is determined by eitherthe length of time necessary to kill (time-dependent) or the effect ofincreasing concentrations (concentration-dependent). To predict thetherapeutic efficacy of antimicrobials with diverse mechanisms of actiondifferent PK/PD parameters may be used. PK/PD parameters may be used todetermine the bioavailability of antimicrobial compositions, forexample, bioavailability of a composition in the pulmonary system,and/or bioavailability of a composition in plasma/serum.

“AUC/MIC ratio” is one example of a PK/PD parameter. AUC is defined asthe area under the plasma/serum or site-of-infection concentration-timecurve of an antimicrobial agent in vivo (in animal or human). Forexample, the site of infection and/or the site where concentration ismeasured can include portions of the pulmonary system, such as bronchialfluid and/or sputum. Accordingly, AUC may include serum AUC, andpulmonary AUC. AUC_((0-t)) can include the area under curve for timezero to a specific time ‘t.’ AUC_((0-inf)) can include the area undercurve from time zero to infinity. AUC/MIC ratio is determined bydividing the 24-hour-AUC for an individual antimicrobial by the MIC forthe same antimicrobial determined in vitro. Activity of antimicrobialswith the dose-dependent killing (such as fluoroquinolones) is wellpredicted by the magnitude of the AUC/MIC ratio.

“C_(max):MIC” ratio is another PK:PD parameter. It describes the maximumdrug concentration in plasma or tissue relative to the MIC.Fluoroquinolones and aminoglycosides are examples where C_(max):MIC maypredict in vivo bacterial killing where resistance can be suppressed.

“Time above MIC” (T>MIC) is another PK/PD parameter. It is expressed apercentage of a dosage interval in which the plasma or site-of-infectionlevel exceeds the MIC. Activity of antimicrobials with thetime-dependent killing (such as beta-lactams or oxazolidinones) is wellpredicted by the magnitude of the T>MIC ratio.

The term “dosing interval” refers to the time between administrations ofthe two sequential doses of a pharmaceutical's during multiple dosingregimens. For example, in the case of orally administered ciprofloxacin,which is administered twice daily (traditional regimen of 400 mg b.i.d)and orally administered levofloxacin, which is administered once a day(500 mg or 750 mg q.d.), the dosing intervals are 12 hours and 24 hours,respectively.

As used herein, the “peak period” of a pharmaceutical's in vivoconcentration is defined as that time of the pharmaceutical dosinginterval when the pharmaceutical concentration is not less than 50% ofits maximum plasma or site-of-infection concentration. In someembodiments, “peak period” is used to describe an interval ofantimicrobial dosing.

The “respirable delivered dose” is the amount of drug inhaled during theinspiratory phase of the breath simulator that is equal to or less than5 microns using a simulator programmed to the European Standard patternof 15 breaths per minute, with an inspiration to expiration ratio of1:1.

Pharmaceutical Compositions

For purposes of the method described herein, a fluoroquinoloneantimicrobial agent formulated with a divalent or trivalent cationhaving improved pulmonary bioavailability may be administered using aninhaler. In some embodiments, a fluoroquinolone antimicrobial disclosedherein is produced as a pharmaceutical composition suitable for aerosolformation, good taste, storage stability, and patient safety andtolerability. In some embodiments, the isoform content of themanufactured fluoroquinolone may be optimized for tolerability,antimicrobial activity and stability.

Formulations can include a divalent or trivalent cation. The divalent ortrivalent cation can include, for example, magnesium, calcium, zinc,copper, aluminum, and iron. In some embodiments, the solution comprisesmagnesium chloride, magnesium sulfate, zinc chloride, or copperchloride. In some embodiments, the divalent or trivalent cationconcentration can be from about 25 mM to about 400 mM, from about 50 mMto about 400 mM, from about 100 mM to about 300 mM, from about 100 mM toabout 250 mM, from about 125 mM to about 250 mM, from about 150 mM toabout 250 mM, from about 175 mM to about 225 mM, from about 180 mM toabout 220 mM, and from about 190 mM to about 210 mM. In someembodiments, the magnesium chloride, magnesium sulfate, zinc chloride,or copper chloride can have a concentration from about 5% to about 25%,from about 10% to about 200/%, and from about 15% to about 20%. In someembodiments, the ratio of fluoroquinolone to divalent or trivalentcation can be 1:1 to 2:1 or 1:1 to 1:2.

Non-limiting fluoroquinolones for use as described herein includelevofloxacin, ofloxacin, ciprofloxacin, enoxacin, gatifloxacin,gemifloxacin, lomefloxacin, moxifloxacin, norfloxacin, pefloxacin,sparfloxacin, garenoxacin, sitafloxacin, and DX-619.

The formulation can have a fluoroquinolone concentration, for example,levofloxacin or ofloxacin, greater than about 50 mg/ml, about 60 mg/ml,about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about110 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190mg/ml, and about 200 mg/ml. In some embodiments, the formulation canhave a fluoroquinolone concentration, for example, levofloxacin orofloxacin, from about 50 mg/ml to about 200 mg/ml, from about 75 mg/mlto about 150 mg/ml, from about 80 mg/ml to about 125 mg/ml, from about80 mg/ml to about 120 mg/ml, from about 90 mg/ml to about 125 mg/ml,from about 90 mg/ml to about 120 mg/ml, and from about 90 mg/ml to about110 mg/ml.

The formulation can have an osmolality from about 300 mOsmol/kg to about500 mOsmol/kg, from about 325 mOsmol/kg to about 450 mOsmol/kg, fromabout 350 mOsmol/kg to about 425 mOsmol/kg, and from about 350 mOsmol/kgto about 400 mOsmol/kg. In some embodiments, the osmolality of theformulation is greater than about 300 mOsmol/kg, about 325 mOsmol/kg,about 350 mOsmol/kg, about 375 mOsmol/kg, about 400 mOsmol/kg, about 425mOsmol/kg, about 450 mOsmol/kg, about 475 mOsmol/kg, and about 500mOsmol/kg.

The formulation can have a pH from about 4.5 to about 8.5, from about5.0 to about 8.0, from about 5.0 to about 7.0, from about 5.0 to about6.5, from about 5.5 to about 6.5, and from 6.0 to about 6.5.

The formulation can comprise a conventional pharmaceutical carrier,excipient or the like (e.g., mannitol, lactose, starch, magnesiumstearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose,glucose, gelatin, sucrose, magnesium carbonate, and the like), orauxiliary substances such as wetting agents, emulsifying agents,solubilizing agents, pH buffering agents and the like (e.g., sodiumacetate, sodium citrate, cyclodextrine derivatives, sorbitanmonolaurate, triethanolamine acetate, triethanolamine oleate, and thelike). In some embodiments, the formulation can lack a conventionalpharmaceutical carrier, excipient or the like. Some embodiments includea formulation lacking lactose. Some embodiments comprise lactose at aconcentration less than about 10%, 5%, 1%, or 0.1%. In some embodiments,the formulation can consist essentially of levofloxacin or ofloxacin anda divalent or trivalent cation.

In some embodiments, a formulation can comprise a levofloxacinconcentration between about 75 mg/ml to about 150 mg/ml, a magnesiumchloride concentration between about 150 mM to about 250 mM, a pHbetween about 5 to about 7; an osmolality of between about 300 mOsmol/kgto about 500 mOsmol/kg, and lacks lactose.

In some embodiments, a formulation comprises a levofloxacinconcentration about 100 mg/ml, a magnesium chloride concentration about200 mM, a pH about 6.2 an osmolality about 383 mOsmol/kg, and lackslactose. In some embodiments, a formulation consists essentially of alevofloxacin concentration about 100 mg/ml, a magnesium chlorideconcentration about 200 mM, a pH about 6.2 an osmolality about 383mOsmol/kg, and lacks lactose. In some embodiments, a formulationconsists of a levofloxacin concentration about 100 mg/ml, a magnesiumchloride concentration about 200 mM, a pH about 6.2 an osmolality about383 mOsmol/kg, and lacks lactose.

In some embodiments, the aerosol fluoroquinolone therapy may beadministered as a treatment or prophylaxis in combination or alternatingtherapeutic sequence with other aerosol, oral or parenteral antibiotics.By non-limiting example this may include tobramycin and/or otheraminoglycoside, aztreonam, carumonam and tigemonam and/or other beta ormono-bactam, ciprofloxacin and/or other fluoroquinolones, azithromycinand/or other macrolides or ketolides, tetracycline and/or othertetracyclines, quinupristin and/or other streptogramins, linezolidand/or other oxazolidinones, vancomycin and/or other glycopeptides, andchloramphenicol and/or other phenicols, and colisitin and/or otherpolymyxins. In more embodiments, the antibiotic can include quinolones,tetracyclines, glycopeptides, aminoglycosides, β-lactams, rifamycins,macrolides/ketolides, oxazolidinones, coumermycins, chloramphenicol,streptogramins, trimethoprim, sulfamethoxazole, or polymyxins. Inparticular embodiments, an antibiotic of the above classes can be, forexample, one of the following. In some embodiments, any of the foregoingantibiotics can be administered by any acceptable method or route, forexample, by aerosol, orally or parenterally.

Beta-Lactam Antibiotics

Beta-lactam antibiotics include, but are not limited to, imipenem,meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine,cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid,cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole,cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefteram, ceftezole,ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam,cephaacetrile, cephalexin, cephaloglycin, cephaloridine, cephalothin,cephapirin, cephradine, cefmetazole, cefoxitin, cefotetan, azthreonam,carumonam, flomoxef, moxalactam, amidinocillin, amoxicillin, ampicillin,azlocillin, carbenicillin, benzylpenicillin, carfecillin, cloxacillin,dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin,penicillin G, piperacillin, sulbenicillin, temocillin, ticarcillin,cefditoren, SC004, KY-020, cefdinir, ceftibuten, FK-312, S-1090,CP-0467, BK-218, FK-037, DQ-2556, FK-518, cefozopran, ME1228, KP-736,CP-6232, Ro 09-1227, OPC-20000, and LY206763.

Macrolides

Macrolides include, but are not limited to, azithromycin,clarithromycin, erythromycin, oleandomycin, rokitamycin, rosaramicin,roxithromycin, and troleandomycin.

Ketolides

Ketolides include, but are not limited to, telithromycin andcethrimycin.

Quinolones

Quinolones include, but are not limited to, amifloxacin, cinoxacin,ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixicacid, norfloxacin, ofloxacin, levofloxacin, oxolinic acid, pefloxacin,rosoxacin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin,moxifloxacin; gemifloxacin; garenofloxacin; PD131628, PD138312,PD140248, Q-35, AM-1155, NM394, T-3761, rufloxacin, OPC-17116, DU-6859a(see, e.g., Sato, K. et al., 1992, Antimicrob Agents Chemother.37:1491-98), and DV-7751a (see, e.g., Tanaka, M. et al., 1992,Antimicrob. Agents Chemother. 37:2212-18).

Tetracyclines, Glycylcyclines and Oxazolidinones

Tetracyclines, glycylcyclines, and oxazolidinones include, but are notlimited to, chlortetracycline, demeclocycline, doxycycline, lymecycline,methacycline, minocycline, oxytetracycline, tetracycline, tigecycline,linezolide, and eperozolid.

Aminoglycosides

Aminoglycosides include, but are not limited to amikacin, arbekacin,butirosin, dibekacin, fortimicins, gentamicin, kanamycin, meomycin,netilmicin, ribostamycin, sisomicin, spectinomycin, streptomycin, andtobramycin.

Lincosamides

Lincosamides include, but are not limited to, clindamycin andlincomycin.

Streptogramins

Streptogramins include, but are not limited to quinupristin.

Glycopeptides

Glycopeptides include, but are not limited to vancomycin.

Polymyxins

Polymyxins include but are not limited to colisitin.

More examples include fosfomycin, penicillins, cephalosporins,carbapenems, penems, and carbacephems.

In some embodiments, a formulation can include a fluoroquinolone incombination with an additional active agent. As discussed herein, somesuch additional active agents can include antibiotics. More additionalactive agents can include bronchodilators, anticholinergics,glucocorticoids, eicosanoid inhibitors, and combinations thereof.Examples of bronchodilators include salbutamol, levosalbuterol,terbutaline, fenoterol, terbutlaine, pirbuterol, procaterol, bitolterol,rimiterol, carbuterol, tulobuterol, reproterol, salmeterol, formoterol,arformoterol, bambuterol, clenbuterol, indacterol, theophylline,roflumilast, cilomilast. Examples of anticholinergics includepratropium, and tiotropium. Examples of glucocorticoids includeprednisone, fluticasone, budesonide, mometasone, ciclesonide, andbeclomethasone. Examples of eicosanoids include montelukast, pranlukast,zafirlukast, zileuton, ramatroban, and seratrodast. More additionalactive agents can include pulmozyme, hypertonic saline, agents thatrestore chloride channel function in CF, inhaled beta-agonists, inhaledantimuscarinic agents, inhaled corticosteroids, and inhaledphosphodiesterase inhibitors. In some embodiments, the aerosolantibiotic therapy administered as a treatment or prophylaxis may beused in combination or alternating therapeutic sequence with anadditional active agent. In more embodiments, the additional activeagent may be administered as a treatment, alone, co-formulated, oradministered with the aerosol antibiotic therapy.

Administration

The fluoroquinolone antimicrobials formulated with divalent or trivalentcations may be administered at a therapeutically effective dosage, e.g.,a dosage sufficient to achieve the outcomes described herein. Similarly,the manner and schedule of administration may be selected to achieve theoutcomes described herein. For example, in some embodiments, therespirable dose administered to the lungs can be from about 20 mg toabout 170 mg, from about 30 mg to about 160 mg, from about 40 mg toabout 150 mg, from about 50 mg to about 140 mg, and from about 65 mg toabout 130 mg. In some embodiments, the loaded dose can be from about 80mg to about 280 mg from about 90 mg to about 270 mg from about 100 mg toabout 260 mg from about 110 mg to about 250 mg, and from about 120 mg toabout 240 mg. In particular embodiments a dose can be administereddaily, or twice daily. In some embodiments, therapy is administered forat least 28 days.

Administration of the fluoroquinolone antimicrobial agents disclosedherein or the pharmaceutically acceptable salts thereof can be via anyof the accepted modes of administration for agents that serve similarutilities including, but not limited to, aerosol inhalation. Methods,devices and compositions for delivery are described in U.S. PatentApplication Publication No. 2006-0276483, incorporated by reference inits entirety.

Pharmaceutically acceptable compositions include solid, semi-solid,liquid and aerosol dosage forms, such as, for example, powders, liquids,suspensions, complexations, liposomes, particulates, or the like.Preferably, the compositions are provided in unit dosage forms suitablefor single administration of a precise dose.

The fluoroquinolone antimicrobial agent can be administered either aloneor in some alternatives, in combination with a conventionalpharmaceutical carrier, excipient or the like (e.g., mannitol, lactose,starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodiumcrosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and thelike). If desired, the pharmaceutical composition can also contain minoramounts of nontoxic auxiliary substances such as wetting agents,emulsifying agents, solubilizing agents, pH buffering agents and thelike (e.g., sodium acetate, sodium citrate, cyclodextrine derivatives,sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate,and the like). Generally, depending on the intended mode ofadministration, the pharmaceutical formulation will contain about 0.005%to 95%, preferably about 0.5% to 50% by weight of a compound of theinvention. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in this art; for example, seeRemington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa.

In one preferred embodiment, the compositions will take the form of aunit dosage form such as vial containing a liquid, solid to besuspended, dry powder, lyophilate, or other composition and thus thecomposition may contain, along with the active ingredient, a diluentsuch as lactose, sucrose, dicalcium phosphate, or the like; a lubricantsuch as magnesium stearate or the like; and a binder such as starch, gumacacia, polyvinylpyrrolidine, gelatin, cellulose, cellulose derivativesor the like.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active compound as definedabove and optional pharmaceutical adjuvants in a carrier (e.g., water,saline, aqueous dextrose, glycerol, glycols, ethanol or the like) toform a solution or suspension. Solutions to be aerosolized can beprepared in conventional forms, either as liquid solutions orsuspensions, as emulsions, or in solid forms suitable for dissolution orsuspension in liquid prior to aerosol production and inhalation. Thepercentage of active compound contained in such aerosol compositions ishighly dependent on the specific nature thereof, as well as the activityof the compound and the needs of the subject. However, percentages ofactive ingredient of 0.01% to 90% in solution are employable, and willbe higher if the composition is a solid, which will be subsequentlydiluted to the above percentages. In some embodiments, the compositionwill comprise 1.0%-50.0 of the active agent in solution.

In some embodiments of the methods, compositions, and uses providedherein, the duration of a therapy, e.g., an aerosolized antibiotictherapy can include at least about 1 day/month, at least about 2days/month, at least about 3 days/month, at least about 4 days/month, atleast about 5 days/month, at least about 6 days/month, at least about 7days/month, at least about 8 days/month, at least about 9 days/month, atleast about 10 days/month, at least about 11 days/month, at least about12 days/month, at least about 13 days/month, at least about 14days/month, at least about 15 days/month, at least about 16 days/month,at least about 17 days/month, at least about 18 days/month, at leastabout 19 days/month, at least about 20 days/month, at least about 21days/month, at least about 22 days/month, at least about 23 days/month,at least about 24 days/month, at least about 25 days/month, at leastabout 26 days/month, at least about 27 days/month, at least about 28days/month, at least about 29 days/month, at least about 30 days/month,and at least about 31 days/month.

Compositions described herein can be administered with a frequency ofabout 1, 2, 3, 4, or more times daily, 1, 2, 3, 4, 5, 6, 7 or more timesweekly, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times monthly. Inparticular embodiments, the compositions are administered twice daily.

Aerosol Delivery

For pulmonary administration, the upper airways are avoided in favor ofthe middle and lower airways. Pulmonary drug delivery may beaccomplished by inhalation of an aerosol through the mouth and throat.Particles having a mass median aerodynamic diameter (MMAD) of greaterthan about 5 microns generally do not reach the lung; instead, they tendto impact the back of the throat and are swallowed and possibly orallyabsorbed. Particles having diameters of about 2 to about 5 microns aresmall enough to reach the upper- to mid-pulmonary region (conductingairways), but are too large to reach the alveoli. Smaller particles,i.e., about 0.5 to about 2 microns, are capable of reaching the alveolarregion. Particles having diameters smaller than about 0.5 microns canalso be deposited in the alveolar region by sedimentation, although verysmall particles may be exhaled.

In one embodiment, a nebulizer is selected on the basis of allowing theformation of an aerosol of a fluoroquinolone antimicrobial agentdisclosed herein having an MMAD predominantly between about 2 to about 5microns. In one embodiment, the delivered amount of fluoroquinoloneantimicrobial agent provides a therapeutic effect for respiratoryinfections. The nebulizer can deliver an aerosol comprising a massmedian aerodynamic diameter from about 2 microns to about 5 microns witha geometric standard deviation less than or equal to about 2.5 microns,a mass median aerodynamic diameter from about 2.5 microns to about 4.5microns with a geometric standard deviation less than or equal to about1.8 microns, and a mass median aerodynamic diameter from about 2.8microns to about 4.3 microns with a geometric standard deviation lessthan or equal to about 2 microns. In some embodiments, the aerosol canbe produced using a vibrating mesh nebulizer. An example of a vibratingmesh nebulizer includes the PART E-FLOW® nebulizer. More examples ofnebulizers are provided in U.S. Pat. Nos. 4,268,460; 4,253,468; 046,146;3,826,255; 4,649,911; 4,510,929; 4,624,251; 5,164,740; 5,586,550;5,758,637; 6,644,304; 6,338,443; 5,906,202; 5,934,272; 5,960,792;5,971,951; 6,070,575; 6,192,876; 6,230,706; 6,349,719; 6,367,470;6,543,442; 6,584,971; 6,601,581; 4,263,907; 5,709,202; 5,823,179;6,192,876; 6,644,304; 5,549,102; 6,083,922; 6,161,536; 6,264,922;6,557,549; and 6,612,303 all of which are hereby incorporated byreference in their entireties. More commercial examples of nebulizersthat can be used with the formulations described herein includeRespirgard II®, Aeroneb®, Aeroneb® Pro, and Aeroneb® Go produced byAerogen; AERx® and AERx® Essence™ produced by Aradigm; Porta-Neb®,Freeway Freedom™, Sidestream Ventstream and I-neb produced byRespironics, Inc.; and PARI LC-Plus®, PARI LC-Star®, produced by PART,GmbH. By further non-limiting example, U.S. Pat. No. 6,196,219, ishereby incorporated by reference in its entirety.

The amount of levofloxacin or ofloxacin that can be administered (as arespirable dose, nebulizer loaded dose, and/or deposited dose) caninclude at least about 20 mg, about 30 mg, about 40 mg, about 50 mg,about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about110 mg, about 120 mg, about 125 mg, about 130 mg, about 140 mg, about150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about350 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, about700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about750 mg, about 760 mg, about 770 mg, about 780 mg, about 790 mg, andabout 800 mg.

The aerosol can be administered to the lungs in less than about 10minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2minutes, and about 1 minute.

Indications

Methods and compositions described herein can be used to treat pulmonaryinfections and disorders, for example, cystic fibrosis. Some embodimentsinclude treating an infection comprising one or more bacteria that caninclude Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonasacidovorans, Pseudomonas alcaligenes, Pseudomonas putida,Stenotrophomonas sp., e.g., Stenotrophomonas maltophilia, Aeromonashydrophilia, Escherichia coli, Citrobacter freundii, Salmonellatyphimurium, Salmonella typhi, Salmonella paratyphi, Salmonellaenteritidis. Shigella dysenteriae. Shigella flexneri, Shigella sonnei,Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae,Klebsiella oxytoca, Serratia marcescens, Morganella morganii, Proteusmirabilis, Proteus vulgaris, Providencia alcalifaciens, Providenciarettgeri, Providencia stuartii, Acinetobacter calcoaceticus,Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis,Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis,Bordetella parapertussis, Bordetella bronchiseptica, Haemophilusinfluenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus,Haemophilus parahaemolyticus, Haemophilus ducreyi. Pasteurellamultocida, Pasteurella haemolytica, Helicobacter pylori, Campylobacterfetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi,Vibrio cholera, Vibrio parahaemolyticus, Legionella pneumophila,Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis,Burkholderia sp., e.g., Burkholderia cepacia, Francisella tularensis,Kingella, and Moraxella. In some embodiments, the pulmonary infectioncan include a gram-negative anaerobic bacteria. In more embodiments, thepulmonary infection can include one or more of the bacteria selectedfrom the group consisting of Bacteroides fragilis, Bacteroidesdistasonis, Bacteroides 3452A homology group, Bacteroides vulgatus,Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis,Bacteroides eggerthii, and Bacteroides splanchnicus. In someembodiments, the pulmonary infection can include a gram-positivebacteria. In some embodiments, the pulmonary infection can include oneor more of the bacteria selected from the group consisting ofCorynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcuspneumoniae, Streptococcus agalactiae, Streptococcus pyogenes,Streptococcus milleri; Streptococcus (Group G); Streptococcus (GroupC/F); Enterococcus faecalis, Enterococcus faecium, Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,Staphylococcus intermedius. Staphylococcus hyicus subsP. hyicus,Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcussaccharolyticus. In some embodiments, the pulmonary infection caninclude a gram-positive anaerobic bacteria. In some embodiments, thepulmonary infection can include one or more bacteria selected from thegroup consisting of Clostridium difficile, Clostridium perfringens,Clostridium tetini, and Clostridium botulinum. In some embodiments, thepulmonary infection can include an acid-fast bacteria. In someembodiments, the pulmonary infection can include one or more bacteriaselected from the group consisting of Mycobacterium tuberculosis,Mycobacterium avium, Mycobacterium intracellulare, and Mycobacteriumleprae. In some embodiments, the pulmonary infection can include anatypical bacteria. In some embodiments, the pulmonary infection caninclude one or more bacteria selected from the group consisting ofChlamydia pneumoniae and Mycoplasma pneumoniae. In some embodiments, thepulmonary infection can comprise a non-fermenting gram-negative bacteria(NFGNB). Examples of NFGNB can include Burkholeria spp.,stenotrophomonas spp., acinetobacter spp., pseudomonas spp., andAchromobacter spp. More example of bacteria useful in the methods andcompositions provided herein can be found in ‘Bergey's Manual ofSystematic Bacteriology,’ Editor-in-chief: Garrity, George M. Boone,David R.; Castenholz, Richard W. (Eds.) Originally published by Williams& Wilkins, 1984, 2nd ed., (2001), incorporated by reference in itsentirety.

Some methods and compositions provided here can be especiallyappropriate for the treatment of pulmonary infections and disorders thatinclude microbial strains that can be difficult to treat using anantimicrobial agent delivered parenterally due to the need for highparenteral dose levels, which can cause undesirable side effects, or dueto lack of any clinically effective antimicrobial agents. For example,administration of an aerosol fluoroquinolone antimicrobial directly tothe site of infection may reduce systemic exposure and can maximize theamount of antimicrobial agent to the site of microbial infection. Suchmethods can be appropriate for treating infections involving microbesthat are susceptible to fluoroquinolone antimicrobials as a way ofreducing the frequency of selection of resistant microbes.

In some embodiments, the aerosol fluoroquinolone antimicrobial agentformulated with divalent or trivalent cations is administered at a levelsufficient to overcome the emergence resistance in bacteria or increasekilling efficiency such that resistance does not have the opportunity todevelop.

Some embodiments of the methods and compositions described hereininclude achieving a reduction in a pulmonary infection. A reduction in apulmonary infection can be measured using a variety of differentmethods. For example, in a pulmonary infection comprising one or moreorganisms, a reduction in the density of the organism can be measured.In some embodiments, treatment can achieve a reduction in the density ofan organism by at least about 1%, about 5%, about 10%, about 15%, about20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 95%, and about 100%. In some embodiments,treatment can achieve a reduction in the density of an organism by atleast about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, and about 100%.

The density of an organism can be measured in a sample taken from asubject, for example, bronchial alveolar lavage, sputum, and serum. Insome embodiments the density of an organism can be reduced by at leastabout 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about0.7, about 0.8, about 0.8, about 1.0, about 1.1, about 1.2, about 1.3,about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5 log₁₀ CFU/gsputum, or more.

Some embodiments of the methods and compositions described herein caninclude achieving an improvement in a pulmonary function parameter.Examples of such parameters can include FEV (forced expiratory volume),FEV₁ (forced expiratory volume in 1 second), and FEF 25-75 (forcedexpiratory flow 25-75%). In some embodiments, the FEV₁ of a subject canbe increased using the methods and compositions described herein, by atleast about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, and more. In someembodiments, the FEVI of a subject can be increased using the methodsand compositions described herein, by at least about 0.01 L, 0.02 L,0.03 L, 0.04 L, and 0.05 L, and by at least about 0.1 L, 0.2 L, 0.3 L,0.4 L, 0.5 L, 0.6 L, 0.7 L, 0.8 L, 0.9 L, 1.0 L, and more.

In some embodiments, the FEF 25-75 of a subject can be increased usingthe methods and compositions described herein, by at least about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, and 25%. In some embodiments, the FEF25-75 of a subject can be increased using the methods and compositionsdescribed herein, by at least about 0.01 L, 0.02 L, 0.03 L, 0.04 L, and0.05 L, and by at least about 0.1 L, 0.2 L, 0.3 L, 0.4 L, 0.5 L, 0.6 L,0.7 L, 0.8 L, 0.9 L, 1.0 L, and more.

Some embodiments of the methods and compositions described herein caninclude reducing the need for a subject to need other inhaled orsystemic antibiotics, such as anti-pseudomonal antimicrobials. Such areduction can be measured by a variety of methods, for example, by theincrease in time to need other inhaled or systemic antibiotics. Areduction in such a need can be measured by a variety of statisticalmeans. For example, hazard ratios may be used in a survival analysis. Insome embodiments, the hazard ratio is less than about 1.0, 0.9, 0.8,0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, and less.

Some embodiments of the methods and compositions described herein caninclude decreasing the frequency of exacerbations, the severity ofexacerbations, the duration of exacerbations, or the likelihood that anexacerbation will occur. An exacerbation can be defined by any ofseveral methods and criteria provided by such methods. In someembodiments, a patient can concurrently meet at least 4 symptoms/signsof the Fuchs definition of an exacerbation (Fuchs H J, et al. Effect ofaerosolized recombinant human DNase on exacerbations of respiratorysymptoms and on pulmonary function in patients with cystic fibrosis. NEngl J Med 1994; 331:637-642, incorporated by reference in itsentirety). The symptoms/signs defined by the Fuchs criteria include:change in sputum; new or increased hemoptysis; increased cough;increased dyspnea; malaise, fatigue or lethargy; temperature above 38°C.; anorexia or weight loss; sinus pain or tenderness; change in sinusdischarge; change in physical examination of the chest; decrease inpulmonary function by 10% or more from a previously recorded value; andradiographic changes indicative of pulmonary infection

In some embodiments, a patient with an improved exacerbation profile canhave at least 1, at least 2, at least 3, and at least 4 of the followingsigns/symptoms, where changes can be relative to a patient's typicalexperience, for example daily experience, and weekly experience. (1)Change in sputum, e.g., for sputum production: patients have no change,a little less or much less amounts of sputum when coughing up, or forchange in sputum appearance: for sputum thickness, patients have alittle thinner or much thinner sputum; for sputum color, patients have abetter color of sputum (better increases frombrown->green->yellow->clear). (2) Hemoptysis, e.g., patients have alittle decrease or a large decrease in the amount of blood coughed up.(3) Cough, e.g., for intensity of cough, patients have a little lighter,or much lighter coughs; for frequency of cough, patients cough a littleless often or much less often. (4) Dyspnea, e.g., for dyspnea withexertion, patients breathe a little easier or much easier whenperforming daily activities. (5) Malaise, fatigue or lethargy, e.g.,patients have a little more energy or much more energy, and/or patientsperform daily activities, e.g., climbing stairs, a little easier, ormuch easier. (6) Temperature, e.g., patients have normal healthytemperature e.g., about 37° C., or patients have no recent history offever. (7) Anorexia or weight loss, e.g., patients have no change inweight, or a little weight gain, and/or patients have a little increasein appetite (8) Sinus pain or tenderness, e.g., patient has no sinuspain or tenderness, or less sinus pain or tenderness. (9) Change insinus discharge, e.g., patients have better sinus discharge (a decreasein thickness and/or better color). (10) Change in physical examinationof the chest, e.g., patients have improved signs on examination of thechest and may report for example, a little decrease chest congestion, ora large decrease in chest congestion. (11) Pulmonary function by 10% ormore from a previously recorded value, e.g., patients have improvedpulmonary function in pulmonary function tests. (12) Radiographicchanges indicative of pulmonary infection, e.g. patients show improvedradiographic changes indicating reduced pulmonary infection.

In some embodiments, exercise tolerance and/or absenteeism fromscheduled events, e.g., school or work can be measured as signs/symptomsof exacerbations.

Table 1 summarizes a variety of methods useful to measure exacerbations.

TABLE 1 Method/Protocol Sign/ Rosenfeld Symptom Fuchs Ramsey CFF(Seattle)^(A) Kraynack^(B) Rabin Blummer Cough X X X X X X X Sputum X XX X X X X Hemoptysis X X X Dyspnea X X X Exercise X X X X ToleranceAbsenteeism X X X X Fatigue X X X Fever X X X X Decreased/Loss X X X X XX Appetite Sinus pain X X Sinus discharge X Chest exam X X X X X XTachypnea X X X Lung function X X X (X) X X X CXR X X X X SaO2 X XNeutrophila X Criteria 4/12 plus ⅖ plus 3/11 Score Score ¾ ¾ antibiotics⅓ ^(A)Rosenfeld M. et al., Defining a pulmonary exacerbation in cysticfibrosis. J. of Pediatrics 139:359-365(2001), incorporated by referencein its entirety. ^(B)Kraynack N. C. et al., Improving care at cysticfibrosis centers through quality improvement. Semin Respir Crit CareMed. 2009 October; 30(5):547-58), incorporated by reference in itsentirety.

Some embodiments of any of the above methods include administeringlevofloxacin or ofloxacin in combination with a divalent or trivalentcation in a dosage amount, administration schedule, and/or method ofadministration sufficient to achieve the above recited outcomes.

Pediatric Patient Populations

Some embodiments provided herein relate to the use of the compositionsprovided herein to treating cystic fibrosis in a human. In someembodiments, the human is a pediatric patient. In some embodiments, thehuman has an age less than about 18 years, less than about 17 years,less than about 16 years, less than about 15 years, less than about 14years, less than about 13 years, less than about 12 years, less thanabout 11 years, less than about 10 years, less than about 9 years, lessthan about 8 years, less than about 7 years, less than about 6 years,less than about 5 years, less than about 4 years, less than about 3years, less than about 2 years, and less than about 1 year.

Dosages of aerosol therapeutic agents for pediatric subjects can be lessthan the dosage for an adult subject. In some embodiments, dosage can bedetermined, in part, due to the weight of a subject. For example, asubject having a weight from about 14 kg to about 21 kg can receive adose of about 120 mg, a subject having a weight from about 22 kg toabout 30 kg can receive a dose of about 180 mg dose, and a subjecthaving a weight more than about 30 kg can receive a dose of about 240mg. In some embodiments, the aerosol therapeutic agent can beadministered daily, or twice daily. In some embodiments, the aerosoltherapeutic agent can be administered for a period of at least about 1day, 3 days, 5 days, 10 days, 15 days, 20 days, and 30 days. In someembodiments, the aerosol therapeutic agent can be administered for about14 days. In particular embodiments the aerosol therapeutic agent isadministered daily for 14 days.

In some embodiments, the human has a body weight less than about 70 kg,less than about 60 kg, less than about 50 kg, less than about 40 kg,less than about 30 kg, less than about 20 kg, and less than about 10 kg.

In some embodiments, the human has a body surface area less than about1.8 m², less than about 1.6 m², less than about 1.4 m², less than about1.2 m², less than about 1.0 m², less than about 0.8 m², less than about0.6 m², and less than about 0.4 m².

In some embodiments, treating one of the above humans comprisesachieving a dose-normalized serum AUC at least about 5 (ng·h/L)/mg Dose,at least about 10 (ng·h/L)/mg Dose, at least about 20 (ng·h/L)/mg Dose,at least about 40 (ng·h/L)/mg Dose, at least about 60 (ng·h/L)/mg Dose,at least about 80 (ng·h/L)/mg Dose, and at least about 100 (ng·h/L)/mgDose.

In some embodiments, treating one of the above humans comprisesachieving a dose-normalized serum C_(max) greater than about 1 μg/L/mgadministered dose, greater than about 2 μg/L/mg administered dose,greater than about 3 μg/L/mg administered dose, greater than about 4μg/L/mg administered dose, greater than about 5 μg/L/mg administereddose, greater than about 6 μg/L/mg administered dose, greater than about7 μg/L/mg administered dose, greater than about 8 μg/L/mg administereddose, greater than about 9 μg/L/mg administered dose, greater than about10 μg/L/mg administered dose, greater than about 11 μg/L/mg administereddose, greater than about 12 μg/L/mg administered dose, greater thanabout 13 μg/L/mg administered dose, greater than about 14 μg/L/mgadministered dose, greater than about 15 μg/L/mg administered dose, andgreater than about 16 μg/L/mg administered dose.

Some embodiments of any of the above methods for treating the recitedhumans include administering levofloxacin or ofloxacin in combinationwith a divalent or trivalent cation in a dosage amount, administrationschedule, and/or method of administration sufficient to achieve therecited outcomes.

EXAMPLES Example 1-Phase 1b Clinical Study with Levofloxacin

A Phase 1 b single-blind, placebo-control, dose-escalating multicenterstudy was carried out to evaluate the safety, tolerability, andpharmacokinetic (PK) profile of aerosolized levofloxacin administered tostable CF patients. All patients had had P. aeruginosa cultured fromsputum within the previous 24 months and at the screening visit. Studydrug was administered twice daily for up to 14 days at three doses byaerosol using a PART eFlow device. Respirable delivered doses (RDD) wereapproximately 40 mg, 80 mg, and 120 mg per treatment, corresponding toloaded doses of 78 mg, 175 mg, and 260 mg, respectively. Thus, theestimated total daily RDDs were 80 mg, 160 mg, and 240 mg. Study drugswere administered at 30 mg/ml (for 40 mg dose) or 50 mg/ml (for 80 mgand 120 mg doses). Table 2 shows the formulations of the study drugs.

TABLE 2 30 mg/ml 50 mg/ml Levofloxacin Levofloxacin solution solutionPlacebo Levofloxacin, 30 (81.6) 50 (136) 0 mg/ml mM) Magnesium, 1.5 (60)2.4 (100) 2.4 (100) mg/ml (mM) Chloride, mg/ml (mM) 4.3 (120) 7.1 (200)7.1 (200) Lactose, mg/ml (mM) 51.4 (150) 51.4 (150) 51.4 (150) pH 6.36.3 6.5 Osmolality, mOsm/kg 314 400 424

All patients used at least 1 concomitant medication during the study.Concomitant medications are those medications taken after the first doseof Study Drug regardless of medication start date. Concomitantmedications used by at least 20% of patients included Salbutamol,Dornase alfa, Azithromycin, Seretide, Pancrelipase, Adeks, Hypertonicsolutions, and Nortase.

Efficacy Results

Table 3 summarizes the results for pulmonary function tests for FEV₁measurements. The greatest relative improvement in FEV₁ after 7 and 14days of dosing was observed in the 120 mg RDD (260 mg loaded)levofloxacin cohort, with a mean improvement of 14.79% and 17.58%,respectively. A mean improvement of 8.90% persisted over the 2 weeks tothe follow-up visit. In addition, there appeared to be a dose responsein patients receiving levofloxacin with patients in the 80 mg and 120 mgdose groups having an improvement in the FEV₁, while those in the 40-mgdose group did not. All 9 patients in the 120 mg dose group had arelative increase in FEV₁, with 4 of 9 patients having at least a 20%increase.

TABLE 3 Median FEVi change (%) for each Treatment Group 40 mg RDD 80 mgRDD 120 mg. RDD Time point Placebo Levofloxacin LevofloxacinLevofloxacin Day 7 n = 10 n = 10 n = 10 n = 9 5.75 −0.75 6.15 10.20 Day14 n = 8 n = 10 n = 10 n = 9 1.20 −1.55 2.95 16.90

Table 4 show the relative changes from baseline across visits (Day 1, 2,7, 14, and 21) in pulmonary function tests for FEV₁. In Table 3,relative change from baseline (CBG) was calculated as ‘Result’ FEV₁minus ‘Baseline’ FEV₁ divided by ‘Baseline’ FEV₁.

TABLE 4 FEV₁ (L) for each Treatment Group 40 mg RDD 80 mg RDD 120 mg.RDD Placebo Levofloxacin Levofloxacin Levofloxacin Time point N = 10 N =10 N = 10 N = 10 Day 1 Predose Baseline n 10 10 10 10 Mean (SD) 2.62(1.150) 2.26 (0.534) 2.06 (0.649) 2.56 (1.121) Median 2.46 2.30 1.952.23 Minimum, maximum 1.31, 4.90 1.58, 3.50 1.17, 3.19 1.43, 4.71 Day 2Predose Result n 10 10 10 10 Mean (SD) 2.75 (1.310) 2.31 (0.594) 2.10(0.709) 2.73 (1.132) Median 2.43 2.32 2.00 2.38 Minimum, maximum 1.33,5.50 1.57, 3.60 1.13, 3.46 1.40, 4.72 Day 2 Relative CFB (%) n 10 10 1010 Mean (SD) 3.85 (6.627) 1.92 (8.315) 1.57 (5.149) 7.29 (10.081) Median2.00 −0.30 1.90 6.60 Minimum, maximum −7.10, 12.20 −6.30, 21.70 −7.60,8.90   −4.70, 23.90 Day 7 Result n 10 10 10 9 Mean (SD) 2.82 (1.361)2.30 (0.544) 2.26 (0.831) 3.07 (1.220) Median 2.35 2.30 2.08 2.40Minimum, maximum 1.39, 5.20 1.49, 3.48 1.20, 3.79 1.79, 4.99 Day 7Relative CFB (%) n 10 10 10 9 Mean (SD) 5.86 (10.196) 1.60 (6.985) 9.42(18.911) 14.79 (12.865) Median 5.75 −0.75 6.15 10.20 Minimum, maximum−6.50, 29.40 −5.70, 17.90 −7.40, 59.90  3.40, 37.70 Day 14 PredoseResult n 8 10 10 9 Mean (SD) 2.99 (1.272) 2.26 (0.524) 2.09 (0.616) 3.12(1.173) Median 3.11 2.30 2.10 2.57 Minimum, maximum 1.47, 5.20 1.55,3.38 1.19, 3.21 1.74, 4.78 Day 14 Relative CFB (%) n 8 10 10 9 Mean (SD)2.00 (5.529) 0.21 (8.440) 2.69 (10.125) 17.58 (15.089) Median 1.20 −1.552.95 16.90 Minimum, maximum −6.70, 8.80   −8.70, 23.10 −19.40, 19.10   1.50, 41.50 Day 21/ET Result n 10 10 10 10 Mean (SD) 2.75 (1.327) 2.16(0.517) 2.03 (0.632) 2.75 (1.155) Median 2.28 2.07 2.04 2.48 Minimum,maximum 1.17, 5.10 1.46, 3.42 1.06, 2.82 1.49, 4.87 Day 21/ET RelativeCFB (%) n 10 10 10 10 Mean (SD) 3.20 (10.157) −4.25 (7.092) −1.03(8.880) 8.90 (15.789) Median 1.75 −5.80 −3.10 10.10 Minimum, maximum−10.70, 20.60   −12.70, 11.80 −11.60, 12.90   −15.90, 38.70   ET = earlytermination.

Colony-forming units of P aeruginosa at Day 1 were compared withcolony-forming units at Day 7 and 14. Table 5 summarizes the percentagechange in sputum P. aeruginosa (log CFU/g) in treatment groups. Declinesin sputum P. aeruginosa were observed over all days of dosing.

TABLE 5 Median Change in sputum P. aeruginosa (log CFU/g) Time forTreatment Group point Placebo Levofloxacin solution Day 7 0.04 (n = 9)−0.50 (n = 21) Day 14 0.04 (n = 9) −1.23 (n = 21)

Safety Results

No study drug-related serious adverse events were reported. Most adverseevents were mild or moderate in severity and self-limiting. The majorityof adverse events were mild, with taste complaints, cough and headacheas the most commonly observed adverse events. No patients receivinglevofloxacin solution for inhalation met drug intolerability criteria.Adverse events reported in more than 1 patient receiving levofloxacinincluded abdominal pain, cough, disease progression (acuteexacerbation), dysgeusia (bad taste), haemoptysis, headache, nasalcongestion, nasopharyngeal pain (sore throat), respiratory tractcongestion, and wheezing. Table 6 summarizes adverse events observed inmore than one CF patient receiving levofloxacin. Accordingly, theseresults demonstrated the safety and tolerability of levofloxacin withmultiple doses over 14 days.

TABLE 6 Treatment group 40 mg 80 mg 120 mg. Levofloxacin LevofloxacinLevofloxacin Placebo Adverse event (N = 10) (N = 10) (N = 10) (N = 10)Abdominal pain 1 (10%) — 1 (10%) — Alopecia — 1 (10%) — — Blood blister— — 1 (10%) — Breath sounds — 1 (10%) — abnormal Chest discomfort —1(10%) — 1(10%) Cough — 4 (40%) 4 (40%) 1 (10%) Diarrhoea 1 (10%) — — —Disease progression — — 2 (20%) 1 (10%) Drooling 1 (10%) Dysguesia 3(30%) 6 (60%) 5 (50%) 1 (10%) Dyspnoea 1 (10%) — — — Fatigue — 1 (10%) —— Flank pain 1 (10%) — — — Forced expired — 1 (10%) — 1 (10%) volumedecreased Hemoptysis — 1 (10%) 1 (10%) — Headache — 2 (20%) 2 (20%) —Migraine — 1 (10%) — — Nasal congestion — 1 (10%) 1 (10%) — Non-cardiac— 1 (10%) — — chest pain Oral candidiasis — 1 (10%) — — Paraethesia oral— — 1 (10%) — Pharyngolaryngeal — — 2 (20%) — pain Pulmonary function —— 1 (10%) — test decreased Pyrexia — — 1 (10%) — Rash erythematous — — 1(10%) — Respiratory tract — — 2 (20%) — congestion Retching — 1 (10%) —— Rhinorrhoea — 1 (10%) — — Thirst — 1 (10%) — — Upper respiratory 1(10%) — — 1 (10%) tract infection Wheezing 1 (10%) — 1 (10%) —

Example 2-Phase 2 Clinical Study with Levofloxacin

A phase 2, multi-center, randomized, double-blind, placebo-controlledstudy was carried out to evaluate the safety, tolerability and efficacyof three dosage regimens of levofloxacin formulated with MgCl₂administered for 28 days to stable CF patients. The following dosageregimens (as nebulizer loaded doses) were evaluated: 120 mg QD (daily);240 mg QD (daily); and 240 mg BID (twice daily). Based on aerosolcharacterization studies of the 100 mg/ml formulation, these loadeddoses of 120 and 240 mg correspond estimated respirable delivered doses(RDDs) using a PART eFlow nebulizer of about 65 and 130 mg,respectively. The plasma levofloxacin Cm. and AUC with all dosageregimens chosen for this study should provide concentrations inpulmonary tissues well in excess of those associated with bactericidalactivity against CF pathogens (data not shown).

The formulation of levofloxacin solution and placebo is shown in Table7. Study drug was administered by aerosol using the PARI eFlow® devicewith a mesh to deliver a particle size smaller than approximately 3.5μm-4.0 μm.

TABLE 7 Levofloxacin Placebo Levofloxacin, mg/ml (mM) 100 (272) 0 Mg,mg/ml (mM) 5.0 (200) 0 Cl, mg/ml (mM) 14.4 (400) 0 pH 5-7 6-8Osmolality, mOsm/kg 350-500 300-500 Saline — 0.9%

Patient inclusion criteria included: (1) at least 16 years of age; (2)clinically diagnosed with CF; (3) able to elicit an FEV₁≥25% but ≤85%predicted value at Screening; (4) received at least 3 courses of inhaledantimicrobials over the preceding 12 months and had received at least 1course of inhaled tobramycin/(TOB®)/colistin in the 2 months prior toVisit 1 (Day 1), but none in the 28 days prior to Visit 1 (Day 1); (5)had a sputum specimen at Screening positive for P. aeruginosa and ahistory of at least 1 positive sputum culture positive for P. aeruginosawithin the last 18 months; and (6) clinically stable with no significantchanges in health status within the last 30 days.

Patient exclusion criteria included: (1) use of an investigational agentwithin 30 days prior to Visit 1 (Day 1); (2) use of any nebulized orsystemic antibiotics active against P. aeruginosa within 28 days priorto Visit 1 (Day 1), other than maintenance oral azithromycin, which musthave been initiated at least 30 days prior to Visit 1 (Day 1); (3)hypersensitivity to fluoroquinolones or excipients of levofloxacinformulated with MgCl₂; (4) intolerance to bronchodilators or unwillingto use a bronchodilator during the study; (5) use of oralcorticosteroids in doses exceeding the equivalent of 10 mgprednisone/day or 20 mg prednisone every other day; (6) changes inphysiotherapy technique or schedule within 14 days prior to Visit 1 (Day1); (7) changes in medical regimen for treatment of CF (e.g.,introduction, dose escalation, or elimination of therapies such asdornase alfa, nonsteroidal anti-inflammatory agents, azithromycin,hypertonic saline, or inhaled corticosteroids) within 30 days of Visit 1(Day 1); (8) history of lung transplantation; (9) evidence of acuteupper respiratory tract infection within 10 days or lower respiratorytract infection within 30 days prior to Visit 1 (Day 1); (10) pregnancy,breastfeeding, or unwilling to practice birth control or abstinenceduring participation in the study (women only); (11) history of seizuresor low seizure threshold (e.g., epilepsy); (12) renal dysfunction(calculated creatinine clearance [CrCl]<50 mL/min) at Screening; (13)aspartate aminotransferase (AST), alanine aminotransferase (ALT), ortotal bilirubin≥3×upper limit of normal (ULN) at Screening or evidenceof severe liver disease (e.g., cirrhosis, portal hypertension); (14)history of human immunodeficiency virus (HIV), hepatitis B, or hepatitisC infection/seropositivity; (15) history of hemoptysis ≥30 mL over any24-hour period during the 30 days prior to Visit 1; (16) oxygensaturation <90% on room air at Screening or Visit 1 (Day 1); and(17)>15% relative decline in FEV₁(L) from Screening to Visit 1 (Day 1).

Patients were assessed on Day 1, 7, 14, and 28, and then up to 28 daysafter completion of study drug, namely, Days 42 and 56. The formulationof study drug, i.e., levofloxacin, is shown in Table 7. The end of thestudy was defined as the last visit of the last patient. Studypopulations included: (1) safety/modified intent to treat (MITT)population which included all patients enrolled in the study thatreceive at least one dose of study drug; (2) efficacy evaluable (EE)population which included all patients enrolled in the study withoutmajor protocol violations that receive at least 80% of study drug dosesduring the 28 days of study drug therapy; and (3) pharmacokinetic (PK)population which included all patients that receive at least one dose ofstudy drug and have at least one PK blood sample collected.

Approximately 32 patients per treatment arm provided 80° % power todetect a difference between treatment arms using a 2-sided analysis ofcovariance (ANCOVA), with a 5% alpha, assuming a standard deviation (SD)of 1.5 and a mean log CFU change in P. aeruginosa of 0.75 decrease, 0.75decrease, no change and 0.25 increase for the levofloxacin 240 mg BID,levofloxacin 240 mg QD, levofloxacin 120 mg QD, and placebo treatmentarms, respectively. Sample size calculations assumed a 10%discontinuation rate from the study; therefore patients who discontinuethe study were not replaced.

Efficacy was evaluated by microbiologic assessment of sputum samples,time to need for anti-pseudomonal antimicrobials, the Cystic FibrosisQuestionaire-Revised (CFQ-R), and pulmonary function tests. In addition,a primary efficacy comparison tested Ho: the average decline in log CFUsof P. aeruginosa from the start of levofloxacin or placeboadministration (Day 1) to four weeks later is equal for all four groups,versus H₁: the average decline in log CFUs is different for at least oneof the four groups using a repeated-measures mixed effects modeladjusting for baseline levofloxacin minimum inhibitory concentration(MIC) as a continuous variable (log base 2 transformed), baseline lungfunction, and geographical region (U.S. versus ex-U.S.). Pair-wisecomparisons between all the treatment arms were conducted as secondaryanalyses.

Efficacy endpoints including clinical endpoints, pulmonary functiontests, and additional microbiological parameters were assessed as changefrom Day 1 to subsequent visits where the endpoint data was collected.Time to need for intravenous/oral/inhaled anti-pseudomonalantimicrobials were assessed from Day 1 until final visit using survivalanalysis. The primary population for efficacy analysis was the EEpopulation, but efficacy endpoints were also analyzed using the MITTpopulation.

Patients enrolled in the study had a median of 5 courses of aerosolizedantibiotics over the previous 12 months. The baseline FEV₁ (as percentpredicted) was 53% across the entire study and not different amonggroups. The median baseline MICs of all isolates of Pseudomonasaeruginosa recovered from all patients (n=592) to levofloxacin was 4mg/L and the MIC90 was 16, also similar across all groups. These MICsare indicative of baseline non-susceptibility/resistance tolevofloxacin, as defined by CLSI reference methods and the US FDA.

Concomitant medication used by patients during the study includedDornase alfa, Azithromycin, Salbutamol, Pancrelipase, hypertonic sodiumchloride, Seretide, and ADEK, and are summarized for the EE populationin Table 8. Table 9 summarizes patient disposition in the study. Theresults of the study showed were statistically significant advantages ofaerosol levofloxacin compared to the placebo in several clinical andmicrobiological measures, despite the use of concomitant medications,resistance to levofloxacin, and previous use of other aerosolizedantibiotics, including tobramycin and colistin.

TABLE 8 Levofloxacin Levofloxacin Levofloxacin Placebo 120 mg QD 240 mgQD 240 mg BID Total Medication (N = 37) (N = 38) (N = 37) (N = 39) (N =151) Dornase alpha 31 (83.8%) 27 (71.1%) 33 (89.2%) 29 (74.4%) 120(79.5%) Azithromycin 25 (67.6%) 29 (76.3%) 26 (70.3%) 32 (82.1%) 112(74.2%) Salbutamol 25 (67.6%) 29 (76.3%) 25 (67.6%) 23 (59.0%) 102(67.5%) Pancrelipase 9 (51.14%) 21 (55.3%) 22 (59.5%) 21 (53.8%) 83(55.0%) Sodium chloride 22 (59.5%) 13 (34.2%) 13 (35.1%) 22 (56.4%) 70(46.4%) Seretide 9 (51.14%) 20 (52.6%) 16 (43.2%) 15 (38.5%) 70 (46.4%)ADEK 10 (27.0%) 13 (34.2%) 10 (27.0%) 13 (33.3%) 46 (30.5%)

TABLE 9 Placebo Levonoxacin Levofloxacin Disposition 120 mg. QD 240 mgQD 240 mg BID Levofloxacin Total Patients Enrolled 37 38 37 39 151Safety/MITT population 37 (100.0%)  38 (100.0%)  37 (100.0%)  39(100.0%) 151 (100.0%) Efficacy Evaluable population 32 (86.5%) 35(92.1%) 35 (94.6%) 34 (87.2%) 136 (90.1%) PK population  37 (100.0%) 37(97.4%)  37 (100.0%)  39 (100.0%) 150 (99.3%) Completed Study 35 (94.6%)37 (97.4%) 35 (94.6%) 36 (92.3%) 143 (94.7%) Discontinued from Study 2(5.4%) 1 (2.6%) 2 (5.4%) 3 (7.7%)  8 (5.3%) Primary Reason Adverse Event2 (5.4%) 1 (2.6%) 1 (2.7%) 2 (5.1%)  6 (4.0%) for Withdrawal of 0 (0.0%)0 (0.0%) 1 (2.7%) 0 (0.0%)  1 (0.7%) Discontinuation consent from StudyOther 0 (0.0%) 0 (0.0%) 0 (0.0%) 1 (2.6%  1 (0.7% Primary Reason forStudy Drug  5 (13.5%) 3 (7.9%) 1 (2.7%) 2 (5.1%) 11 (7.3%)Discontinuation^(a) Adverse Event^(a)  4 (10.8%) 3 (7.9%) 1 (2.7%) 2(5.1%) 10 (6.6%) Other 1 (2.7%) 0 (0.0%) 0 (0.0%) 0 (0.0%)  1 (0.7%)^(a)Patients could be included in both discontinuation from study andStudy Drug categories.

Microbiological Evaluations

Changes in P. aeruginosa density (log lo CFU/g sputum) for sputumsamples taken from the EE and MITT populations are summarized in Tables10 and 11, respectively (SD=standard deviation). Changes in MIC for P.aeruginosa density (log¹⁰ CFU/g sputum) to levofloxacin (all organisms)for EE and MITT populations are shown in Tables 12 and 13, respectively.In addition, changes in the highest MIC values for P. aeruginosaisolates to levofloxacin were determined for EE and MITT populations,and are summarized in Tables 14 and 15, respectively. Tables 16 and 17summarize the percentage of EE and MITT populations, respectively, withcategorical changes in highest MIC values for P. aeruginosa isolates tolevofloxacin. Tables 18 and 19 summarize changes in non-fermenting gramnegative bacilli density (NFGNB), excluding P. aeruginosa, with onlypatients with positive NFGNB counts at baseline, for EE and MITTpopulations, respectively. Tables 20 and 21 summarize changes forpresumptive S. aureus density (log₁₀ CFU/g sputum) for only patientswith positive S. aureus counts at baseline, for EE and MITT populations,respectively.

TABLE 10 EE population—P. aeruginosa density (log₁₀ CFU sputum) CombinedLevofloxacin Levofloxacin Levofloxacin Levofloxacin 240 mg QD Placebo120 mg. QD 240 mg QD 240 mg BID and BID (N = 32) (N = 35) (N = 35) (N =34) (N = 69) Day 1 Mean 7.49   7/43 (1.622)   7.16 (1.597)   7.26(1.433)   7.21 (1.508) (SD) (1.674) Median 8.11 8.17 7.97 7.84 7.90 Day7 Mean 7.37   6.64 (1.818)   6.94 (1.783)   6.23 (1.850)   6.59 (1.838)(SD) (1.746) Median 8.14 6.86 7.15 6.52 6.77 Change Mean −0.12 −0.76(1.263) −0.25 (1.789) −1.03 (1.921) −0.64 (1.833) from (SD) (1.422)Baseline Median 0.09 −0.58 −0.18 −0.88 −0.37 to Day 7 P-value 0.02770.5892 0.0051 0.0533 Day 14 Mean 7.48   7.32 (1.643)   6.69 (1.628)  6.43 (1.725)   6.56 (1.669) (SD) (1.616 Median 7.85 8.00 6.46 6.816.78 Change Mean 0.03 −0.10 (1.240) −0.47 (1.905) −0.83 (1.901) −0.65(1.898) from (SD) (1.355) Baseline Median −0.01 0.00 −0.11 −0.91 −0.46to Day 14 P-value 0.3929 0.0998 0.0047 0.0105 Day 28 Mean 7.85   7.25(1.431)   6.78 (1.896)   6.65 (1.440)   6.72 (1.683) (SD) (1.050) Median8.28 7.78 7.38 6.40 6.90 Change Mean 0.36 −0.31 (1.050) −0.38 (1.780)−0.74 (1.488) −0.55 (1.645) from (SD) (1.329) Baseline Median 0.00 −0.20−0.52 −0.68 −0.58 to Day 28 P-Value 0.0093 0.0075 0.0002 0.0002

TABLE 11 MITT population—P. aeruginosa density (log₁₀ CTU/g sputum)Combined Levofloxacin Levofloxacin Levofloxacin Levofloxacin 240 mg QDPlacebo 120 mg. QD 240 mg QD 240 mg BID and BID (N = 37) (N = 38) (N =37) (N = 39) (N = 76) Day 1 Mean 7.45 7.43 7.08 7.17 7.12 (SD) (1.656)(1.571) (1.742) (1.726) (1.723) Median 8.10 8.16 7.97 7.90 7.91 Day 7Mean 7.21 6.68 6.79 6.16 6.47 (SD) (1.750) (1.778) (1.854) (1.969)(1.927) Median 7.97 6.98 6.95 6.53 6.69 Change Mean −0.24 −0.70 −0.31−0.97 −0.65 from (SD) (1.429) (1.257) (1.823) (1.846) (1.852) BaselineMedian 0.00 −0.53 −0.18 −0.76 −0.35 to Day 7 P-value 0.1091 0.69860.0124 0.0956 Day 14 Mean 7.33 7.33 6.63 6.32 6.48 (SD) (1.651) (1.602)(1.744) (1.875) (1.806) Median 7.85 8.00 6.46 6.70 6.61 Change Mean 0.07−0.07 −0.44 −0.81 −0.63 from (SD) (1.373) (1.213) (1.855) (1.838)(1.843) Baseline Median −0.02 0.00 −0.10 −0.79 −0.40 to Day 14 P-value0.79411 0.1972 0.0091 0.0251 Day 28 Mean 7.66 7.26 6.82 6.58 6.70 (SD)(1.236) (1.394) (1.869) (1.704) (1.781) Median 8.26 7.78 7.38 6.48 6.90Change Mean 0.21 −0.27 −0.25 −0.66 −0.46 from (SD) (1.377) (1.035)(1.857) (1.422) (1.658) Baseline Median 0.00 −0.20 −0.39 −0.56 −0.5 toDay 28 P-Value 0.0728 0.0698 0.0014 0.0039

TABLE 12 EE population—MIC of P. aeruginosa to levofloxacin (allorganisms) (μg/ml) Combined Levofloxacin Placebo LevofloxacinLevofloxacin Levofloxacin 240 mg QD and (N = 37) 120 mg. QD 240 mg QD240 mg BID BID Day 1 N 124 140 140 136 276 Mean (SD) 6.0 (5.00) 11.5(21.98) 6.3 (8.85) 6.8 (8.80)  6.5 (8.82)  MIC₅₀ 4 4 4 4 4 MIC₉₀ 16 3216 16 16 Day 7 N 124 136 132 132 264 Mean (SD) 6.6 (6.73)  9.9 (17.28) 6.8 (16.13) 8.8 (17.28) 7.8 (16.71) Min, Max 0.25, 32 0.13, 128 0.13,128 0.13, 128 0.13, 128 MIC₅₀ 4 4 2 4 4 MIC₉₀ 8 16 16 16 16 Day 14 N 120140 136 132 268 Mean (SD) 5.6 (6.79) 13.0 (29.34) 6.6 (9.44) 8.4 (14.47)7.5 (12.19) MIC₅₀ 4 4 4 4 4 MIC₉₀ 8 37 16 16 16 Day 28 N 128 132 136 124260 Mean (SD) 5.0 (3.97)  8.9 (13.47) 6.0 (7.57) 10.1 (16.32)  8.0(12.67) MIC₅₀ 4 4 4 4 4 MIC₉₀ 8 16 16 32 16

TABLE 13 MITT population—MIC of P. aeruginosa to levofloxacin (allorganisms) (μg/ml) Combined Levotioxacin Levolloxacin LevofloxacinLevotioxacin 240 mg QD r Placebo 120 mg. QD 240 mg QD 240 mg BID and BIDDay 1 N 140 152 148 157 300 Mean (SD) 6.1 (5.40) 11.0 (21.17) 6.1 (8.64)6.8 (8.47)  6.4 (8.55)  MIC₅₀ 4 4 4 4 4 MIC₉₀ 16 32 16 16 16 Day 7 N 136144 140 140 780 Mean (SD) 6.8(7.33)  9.6 (16.83)  6.7 (15.69) 8.6(16.82) 7.6 (16.27) MIC₅₀ 4 4 4 4 4 MIC₉₀ 16 16 16 16 16 Day 14 N 128144 144 140 284 Mean (SD) 5.8 12.9 (28.95) 6.5 (9.23) 8.2 (14.11) 7.3(11.90) MIC₅₀ 4 6 4 4 4 MIC₉₀ 16 32 16 16 16 Day 28 N 137 136 140 137272 Mean (SD) 4.9 (3.97)  8.8 (13.32) 5.9 (750)  9.9 (15.98) 7.9 (12.51)MIC₅₀ 4 4 4 4 4 MIC₉₀ 8 16 16 32 16

TABLE 14 EE population—MIC of P. aeruginosa to levofloxacin (allorganisms) (μg/ml) Combined Levofloxacin Levofloxacin LevofloxacinLevofloxacin 240 mg QD Placebo 120 mg. QD 240 mg QD 240 mg BID and BID(N = 32) (N = 35) (N = 35) (N = 34) (N = 69) Day 1 Mean (SD) 8.79(6.661) 14.98 (22.383) 9.35 (12.991) 11.58 (13.399) 10.46 (13.144) MIC₅₀8 8 4 6 4 MIC₉₀ 16 32 32 32 32 Day 7 Mean (SD) 8.77 (7.455)15.71(22.523) 10.86 (22.222)  13.05 (22.838) 11.95 (22.385) MIC₅₀ 8 8 44 MIC₉₀ 16 32 16 32 32 Day 14 Mean (SD)  8.42 (11.437) 21.16 (43.639)9.56 (12.409) 15.18 (25.541) 12.33 (20.032) MIC₅₀ 6 8 8 4 8 MIC₉₀ 16 3216 32 3 Day 28 Mean (SD) 7.31 (4.961) 12.91 (15.669) 9.42 (11.658) 17.97(26.008) 13.56 (20.252) MIC₅₀ 8 8 8 8 8 MIC₉₀ 16 32 16 32 32

TABLE 15 MITT population—Highest MIC of P. aeruginosa to levofloxacin(μg/ml) Combined Levofloxacin Levofloxacin Levofloxacin Levofloxacin 240mg QD Placebo 120 mg. QD 240 mg QD 240 mg BID and BID (N = 37) (N = 38)(N = 37) (N = 39) (N = 76) Day 1 Mean (SD) 9.04 (7.593) 14.32 (21.583)9.06 (12.685) 11.41 (12.816) 10.25 (12.720  MIC₅₀ 8 8 4 6 4 MIC₉₀ 16 3232 32 32 Day 7 Mean (SD) 9.06 (8.203) 15.17 (21.991) 10.53 (21.615) 12.96 (21.902) 11.76 (21.639) MIC₅₀ 8 8 4 6 4 MIC₉₀ 16 32 16 32 16 Day14 Mean (SD)  8.78 (11.554) 20.34 (42.555) 9.28 (12.136) 14.86 (24.522)12.07 (19.415) MIC₅₀ 6 8 8 6 8 MIC₉₀ 16 32 16 32 32 Day 28 Mean (SD)7.78 (6.334) 12.74 (15.286) 9.17 (11.389) 17.91 (25.067) 13.48 (19.734)MIC₅₀ 8 8 8 8 8 MIC₉₀ 16 32 16 16 32

TABLE 16 EE population— Categorical change in highest MIC of P.aeruginosa to levofloxacin (% population) Combined Levo− Levo− Levo−Levo− floxiacin floxacin floxacin floxacin 240 mg 120 mg. 240 mg 240 mgQD Placebo QD QD BID and BID (N = 32) (N = 35) (N = 35) (N = 34) (N =69) Change >=4 fold  6.5%  2.9% 12.1%  9.1% 10.6% from increase Baseline<4 fold 93.5% 97.1% 87.9% 90.9% 89.4% to Day 7 increase Change >=4 fold10.0% 11.4%  5.9% 18.2% 11.9% from increase Baseline <4 fold 90.0% 88.6%94.1% 81.8% 88.1% to Day 14 increase Change >=4 fold  9.4%  9.1%  8.8%15.6% 10.6% from increase Baseline <4 fold 90.6% 90.9% 91.2% 84.4% 87.9%to Day 28 increase Change >=4 fold 22.6%  6.2%  5.7% 15.2% 10.3% fromincrease Baseline <4 fold 77.4% 93.6% 94.3% 84.8% 89.7% to Day increase56/Early Term

TABLE 17 MITT population—Categorical change in highest MIC of P.aeruginosa to levofloxacin (% population) Combined Levo− Levo− Levo−Levo− floxacin floxacin floxacin floxacin 240 mg 120 mg. 240 mg. 240 mgQD and Placebo QD QD BID BID (N = 37) (N = 38) (N = 37) (N = 39) (N =76) Change >=4 fold  5.7%  2.8% 11.4%  8.3%  9.9% from increase Baseline<4 fold 94.3% 97.2% 88.6% 91.7% 90.1% to Day 7 increase Change >=4 fold 8.8% 10.8%  5.6% 16.7% 11.1% from increase Baseline <4 fold 91.2% 89.2%94.4% 83.3% 88.9% to Day 14 increase Change >=4 fold  8.3%  8.6%  8.3%14.3% 11.3% from increase Baseline <4 fold 91.7% 91.4% 91.7% 85.7% 88.7%to Day 28 increase Change >=4 fold 20.0%  5.9%  5.4% 13.9%  9.6% fromincrease Baseline <4 fold 80.0% 94.1% 94.6% 86.1% 90.4% to Day increase56/Early Term

TABLE 18 EE population—Change in non−fermenting grain negative bacillidensity (log₁₀ CFU/g sputum) Combined Levo- Levo- Levo- Levo- floxacinfloxacin floxacin floxacin 240 mg Combined 120 mg. 240 mg 240 mg QDLevo- Placebo QD QD BID and BID floxacin (N = 5) (N = 5) (N = 5) (N = 4)(N = 9) (N = 14) Day 1 Mean 6.60 5.69 4.65 5.52 5.03 5.27 (SD) (1.113)(2.674) (2.271) (2.291) (2.182) (2.288) Day 7 Mean 6.26 4.54 2.18 5.013.44 3.83 (SD) (3.018) (2.361) (1.614) (3.068) (2.657) (2.522) ChangeMean −0.34 −1.15 −2.47 −0.51 −1.60 −1.44 from (SD) (2.610) (1.607)(3.346) (3.184) (3.235) (2.699) Baseline P- 0.1206 0.0476 0.7552 0.17550.1178 to Day 7 value Day 14 Mean 5.94 3.15 2.04 6.02 3.80 3.57 (SD)(3.002) (3.122) (2.315) (1.894) (2.903) (2.880) Change Mean −0.66 −2.54−2.61 0.50 −1.23 −1.70 from (SD) (2.315) (3.057) (3.222) (1.638) (2.980)(2.961) Baseline P- 0.0894 0.1571 0.5983 0.5932 0.2824 to Day value 28Day 28 Mean 5.60 5.10 1.36 5.01 2.98 3.63 (SD) (3.012) (2.938) (0.808)(2.758) (2.621) (2.787) Change Mean −1.00 −1.44 −3.29 −0.51 −2.05 −1.87from (SD) (3.262) (4.739) (2.401) (1.438) (2.407) (3.092) Baseline P-0.2997 0.0479 0.9881 0.2237 0.1956 to Day value 28

TABLE 19 MITT population—Change in non-fermenting gram negative bacillidensity (log₁₀ CFU/g sputum) Combined Levo- Levo- Levo- Levo- floxacinfloxacin floxacin floxacin 240 mg Combined 120 mg. 240 mg 240 mg QDLevo- Placebo QD QD BID and BID floxacin (N = 6) (N = 5) (N = 7) (N = 5)(N = 12) (N = 17) Day 1 Mean 5.88 5.69 5.26 6.05 5.59 5.62 (SD) (2.018)(2.674) (2.203) (2.317) (2.184) (2.251) Day 7 Mean 5.44 4.54 2.52 5.633.82 4.03 (SD) (3.375) (2.361) (1.997) (3.004) (2.832) (2.650) ChangeMean −0.45 −1.15 −2.73 −0.42 −1.77 −1.59 from (SD) (2.350) (1.607)(2.882) (2.765) (2.955) (2.594) Baseline P-value 0.2680 0.0485 0.65620.3749 0.2748 to Day 7 Day 14 Mean 5.17 3.25 3.08 6.50 4.50 4.11 (SD)(3.286) (3.122) (2.599) (1.964) (2.862) (2.911) Change Mean −0.72 −2.54−2.18 0.45 −1.08 −1.51 from (SD) (2.075) (3.057) (2.784) (1.423) (2.605)(2.733) Baseline P- 0.1020 0.1924 0.2832 0.9333 0.4410 to Day value 28Day 28 Mean 4.88 5.10 2.77 5.63 3.96 4.24 (SD) (3.215) (2.938) (2.505)(2.762) (2.892) (2.850) Change Mean −1.00 −1.44 −2.49 −0.43 1.63 −1.58from (SD) (2.917) (4.739) (2.538) (1.260) (2.284) (2.885) Baseline P-0.5733 0.1606 0.4532 0.7266 0.6211 to Day value 28

TABLE 20 EE population—Change for presumptive S. aureus density (log₁₀CFU/g sputum) Combined Levo- Levo- Levo- Levo- floxacin floxacinfloxacin floxacin 240 mg Combined 120 mg. 240 mg 240 mg QD Levo- PlaceboQD QD BID and BID floxacin (N = 16) (N = 16) (N = 19) (N = 13) (N = 32)(N = 48) Day 1 Mean 5.67 5.72 5.64 5.68 5.65 5.68 (SD) (2.125) (1.871)(1.985) (2.196) (2.038) (1.964) Day 7 Mean 5.53 4.53 5.34 4.96 5.18 4.97(SD) (2.152) (2.272) (2.321) (2.992) (2.574) (2.473) Change Mean −0.14−1.19 −0.30 −0.72 −0.47 −0.71 from (SD) (0.968) (1.722) (1.579) (1.159)(1.419) (1.546) Baseline P- 0.0347 0.5969 0.2039 0.2891 0.1059 to Day 7value Day 14 Mean 5.35 5.30 5.14 4.70 4.96 5.08 (SD) (2.469) (2.543)(2.594) (2.829) (2.656) (2.597) Change Mean −0.32 −0.42 −0.50 −0.98−0.69 −0.60 from (SD) (1.259) (1.273) (1.766) (0.998) (1.502) (1.422)Baseline P- 0.6684 0.5883 0.1593 0.2497 0.3259 to Day value 28 Day 28Mean 5.46 5.31 4.56 5.42 4.89 5.04 (SD) (2.647) (2.482) (2.724) (2.973)(2.806) (2.680) Change Mean −0.21 −0.41 −1.08 −0.38 −0.81 −0.67 from(SD) (1.137) (1.146) (2.622) (1.144) (2.173) (1.882) Baseline P- 0.61240.1186 0.6403 0.2589 0.3159 to Day value 28

TABLE 21 EE population—Change for presumptive S. aureus density (log₁₀CPU/g sputum) Combined Levo- Levo- Levo- Levo- floxacin floxacinfloxacin floxacin 240 mg Combined 120 mg. 240 mg 240 mg QD Levo- PlaceboQD QD BID and BID floxacin (N = 19) (N = 17) (N = 21) (N = 16) (N = 37)(N = 54) Day 1 Mean 6.06 5.89 5.63 5.51 5.58 5.68 (SD) (2.152) (1.941)(1.894) (2.318) (2.058) (2.009) Day 7 Mean 5.78 4.75 5.23 4.65 4.99 4.91(SD) (2.071) (2.382) (2.249) (2.954) (2.543) (2.472) Change Mean −0.28−1.14 −0.40 −066 −0.51 −0.71 from (SD) (0.985) (1.681) (1.615) (1.243)(1.458) (1.545) Baseline P- 0.0563 0.6856 0.3058 0.3995 0.1653 to Day 7value Day 14 Mean 5.64 5.48 4.97 4.32 4.70 4.9:5 (SD) (2.387) (2.568)(2.579) (2.820) (2.662) (2.633) Change Mean −0.42 −0.41 −0.66 −0.99−0.80 −0.68 from (SD) (1.216) (1.233) (1.907) (0.981) (1.578) (1475)Baseline P- 0.8238 0.5691 0.2231 0.2393 0.4058 to Day value 28 Day 28Mean 5.65 5.49 4.48 4.95 4.66 4.93 (SD) (2.511) (2.510) (2.633) (3.018)(2.760) (2.684) Change Mean −0.41 −0.41 −1.15 −0.44 −0.87 −0.72 froin(SD) (1.240) (1.110) (2.578) (1.150) (2.131) (1.860) Baseline P- 0.83410.1623 0.7846 0.3528 0.4572 to Day value 28

P. aeruginosa densities were reduced from baseline values on (Day 1)over the course of the study in all three levofloxacin treatment groupsin both the EE and MITT populations. FIGS. 3 and 4 show mean changes inP. aeruginosa densities over time for each treatment group.

In the EE population, there was a decrease of P. aeruginosa density inpatients administered levofloxacin 240 mg BID from a median value of7.84 log₁₀ CFU/g sputum on Day 1 to a median value of 6.40 log₁₀ CFU/gsputum on Day 28, representing a reduction in P. aeruginosa density insputum of approximately 96%. In patients administered levofloxacin 240mg QD, there was a decrease in P. aeruginosa density from a median valueof 7.97 log₁₀ CFU/g sputum on Day 1 to a median value of 7.38 log₁₀CFU/g sputum on Day 28, representing a reduction in P. aeruginosadensity in sputum of approximately 74%. In patients administeredlevofloxacin 120 mg QD, there was a decrease in P. aeruginosa densityfrom a median value of 8.17 log₁₀ CFU/g sputum on Day 1 to a medianvalue of 7.78 log₁₀ CFU/g sputum on Day 28, representing a reduction inP. aeruginosa density in sputum of approximately 59%. The largestdifferences for P. aeruginosa density in sputum between patientsadministered placebo and patients administered levofloxacin were 1.62log₁₀ CFU/g sputum on Day 7, 1.39 log₁₀ CFU/g sputum on Day 14, and 1.88log₁₀ CFU/g sputum on Day 28.

In the MITT population, there was a decrease of P. aeruginosa density inpatients administered levofloxacin 240 mg BID from a median value of7.90 log₁₀, CFU/g sputum on Day 1 to a median value of 6.48 log₁₀ CFU/gsputum on Day 28, representing a reduction in P. aeruginosa density insputum of approximately 96%. In patients administered levofloxacin 240mg QD, there was a decrease in P. aeruginosa density from a median valueof 7.97 log₁₀ CFU/g sputum on Day 1 to a median value of 7.38 log₁₀CFU/g sputum on Day 28, representing a reduction in P. aeruginosadensity in sputum of approximately 74%. In patients administeredlevofloxacin 120 mg QD, there was a decrease in P. aeruginosa densityfrom a median value of 8.16 log₁₀ CFU/g sputum on Day 1 to a medianvalue of 7.78 log₁₀ CFU/g sputum on Day 28, representing a reduction inP. aeruginosa density in sputum of approximately 58%. The largestdifferences for P. aeruginosa density in sputum between patientsadministered placebo and patients administered levofloxacin were 1.44log₁₀ CFU/g sputum on Day 7, 1.39 log₁₀ CFU/g sputum on Day 14, and 1.78log₁₀ CFU/g sputum on Day 28.

For the EE and MITT populations, baseline MIC50 values for P. aeruginosaisolates from patients administered placebo, 120 mg QD, 240 mg QD, and240 mg BID were 8 μg/ml, 8, 4 μg/ml, and 6 μg/ml, respectively. On Day28, MIC₅₀ values for P. aeruginosa isolates from patients administeredplacebo, 120 mg QD, 240 mg QD, and 240 mg BID were 8 μg/ml, 8 μg/ml, 8μg/ml, and 8 μg/ml, respectively. Baseline MIC₉₀ values for P.aeruginosa isolates from patients administered placebo, 120 mg QD, 240mg QD, and 240 mg BID were 16 μg/ml, 32 μg/ml, 32 μg/ml, and 32 μg/ml,respectively. On Day 28, MIC₉₀ values for P. aeruginosa isolates frompatients administered placebo, 120 mg QD, 240 mg QD, and 240 mg BID were16 μg/ml, 32 μg/ml, 16 μg/ml, and 32 μg/ml, respectively.

The similarity of corresponding MIC₅₀ and MIC₉₀ values between Day 1 andDay 28 for the EE and MITT populations indicates that P. aeruginosacultures from patients did not develop any significant resistance tolevofloxacin.

Clinical Evaluations—CFQ-R Questionnaire

Patients completed the CFQ-R questionnaire with domains that included:respiratory, body image, digestion, eating, emotion, health perception,physical, role/school, social, treatment burden, vitality, and weight.Tables 22 and 23 summarize results for the respiratory domain of theCFQ-R for EE and MITT populations, respectively. Table 24 summarizeschanges in score for various domains of the CFQ-R from baseline to visit4 in the MITT population.

TABLE 22 EE population—Respiratory domain of CFQ-R Com- bined Levo-Levo- Levo- Levo- floxacin Pla- floxacin floxacin floxacin 240 mg cebo120 mg. 240 mg 240 mg QD (N = QD QD BID and BID 32) (N = 35) (N = 35) (N= 34) (N = 69) Day Mean 66.7 63.5 60.8 60.3 60.5 1 (SD) (14.39) (15.84)(15.83) (16.42) (16.00) Median 66.7 66.7 61.1 61.1 61.1 Day Mean 66.767.8 63.2 68.8 66.0 14 (SD) (15.84) (13.79) (19.34) (19.15) (19.31)Median 63.9 66.7 66.7 72.2 66.7 Change Mean −0.0 4.3 2.1 8.5 5.3 from(SD) (11.20) (9.82) (16.41) (155.5) (16.19) Base- Median 0.0 5.5 0.0 5.65.6 line P- 0.3953 0.9534 0.0374 0.2171 to Day value 14 Day Mean 64.965.1 61.7 66.0 63.8 28 (SD) (18.53) (17.28) (19.37) (19.68) (19.49)Median 63.9 66.7 66.7 72.2 66.7 Change Mean −1.7 1.6 1.0 5.2 3.0 from(SD) (14.90) (12.32) (14.91) (17.56) (16.27) Base- Median 0.0 0.0 0.05.5 0.0 line P- 0.6183 0.8145 0.0924 0.2690 to Day value 28

TABLE 23 MITT population—Respiratory domain of CFQ-R Com- bined Levo-Levo- Levo- Levo- floxacin Pla- floxacin floxacin floxacin 240 mg cebo120 mg. 240 mg. 240 mg QD (N = QD QD BID and BID 37) (N = 38) (N = 37)(N = 39) (N = 76) Day 1 Mean 64.9 62.3 61.3 60.5 60.9 (SD) (14.17)(15.88) (15.52) (16.31) (15.83) Median 66.7 61.1 61.1 61.1 61.1 Day 14Mean 64.6 67.1 63.0 68.4 65.8 (SD) (15.84) (13.70) (19.74) (19.51)(19.68) Median 61.1 66.7 66.7 72.2 69.4 Change Mean −0.3 4.5 1.4 7.5 4.5from (SD) (10.79) (9.78) (17.54) (15.30) (16.59) Base- Median 0.0 5.50.0 5.6 5.5 line P- 0.2559 0.8964 0.0334 0.1937 to Day value 14 Day 28Mean 63.1 64.6 61.6 64.4 63.0 (SD) (18.01) (16.94) (19.74) (20.39)(19.98) Median 61.1 66.7 66.7 72.2 66.7 Min, 22, 94 17, 100 17, 89 22,94 17, 94 Max Change Mean −1.8 2.0 0.3 3.0 1.7 from (SD) (14.11) (12.23)(16.14) (19.18) (17.66) Base- Median 0.0 0.0 0.0 0.0 0.0 line P- 0.50290.8365 0.2174 0.4069 to Day value 28

TABLE 24 LS Mean Change from Baseline Placebo Levofloxacin 240 mg. BIDLS Mean Difference CFQ-R Scale (N = 37) (N = 39) (95% CI) P-ValueRespiratory −0.44 4.06 4.50 (−2.68, 11.67) 0.2174 Body Image 0.59 0.34−0.25 (−6.00, 5.50) 0.9315 Digestion −1.35 1.11 2.46 (−3.27, 8.18)0.3975 Eating −3.29 2.41 5.70 (0.78, 10.62) 0.0235 Emotion 2.32 2.22−0.09 (−4.34, 4.15) 0.9651 Health −2.1 0.52 2.58 (−3.70, 8.86) 0.4117Perception Physical −2.80 3.14 5.94 (0.75, 11.13) 0.0252 Role/School−1.41 −1.94 −0.53 (−5.43, 4.38) 0.8324 Social −1.68 0.69 2.37 (−2.66,7.40) 0.3530 Treatment −0.99 −0.93 0.06 (−5.84, 5.95) 0.9851 BurdenVitality −1.88 1.23 3.11 (−2.91, 9.13) 0.3085 Weight 4.72 10.06 5.4(−4.38, 15.06) 0.2792

For the EE population, mean changes from baseline to Day 28 forrespiratory factors measured by the CFQ-R for patient administeredplacebo, 120 mg QD, 240 mg QD, and 240 mg BID treatment groups were 1.6,1.0, 5.2, and 3.0 units, respectively. For the MITT population, meanchanges from baseline to Day 28 for respiratory factors for patientadministered placebo, 120 mg QD, 240 mg QD, and 240 mg BID treatmentgroups were 2.0, 0.3, 3.0, and 1.7 units, respectively. The 240 mg BIDgroup in the EE population demonstrated a statistically significantimprovement in respiratory score on Day 14.

The CFQ-R eating score also showed improvement with the difference inthe 240 mg BID group in the MITT population showing statisticallysignificant improvement at Day 28.

Time to Patient Need for Anti-Pseudomonal Antimicrobials

Time to administration of intravenous/oral/inhaled anti-pseudomonalantimicrobials from Day 1 until Final visit was measured for patientswith at least one of the following: decreased exercise tolerance,increased cough, increased sputum/chest congestion, and decreasedappetite. The proportion of patients requiring anti-pseudomonalantimicrobials (inhaled or systemic) over time was analyzed using a Coxproportional hazards model. Table 25 summarizes results for time to needfor anti-pseudomonal antimicrobials for EE and MITT populations.

TABLE 25 EE and MITT populations—Parameters for time to needanti-pseudomonal antimicrobials Combined Levo- Levo- Levo- Levo-floxacin floxacin floxacin floxacin 240 mg 120 mg. 240 mg 240 mg QDPlacebo QD QD BID and BID (N = 32) (N = 35) (N = 35) (N = 34) (N = 69)EE Patients 40.6% 20.0% 25.7% 20.6% 23.2% popu- Requiring lation Anti-Pseudo- monal Anti- microbials Hazard 0.39 0.48 0.26 0.37 Ratio [0.15,[0.20, [0.10, [0.17, [95% CI] 1.01] 1.17] 0.69] 0.81] P-value 0.05220.1050 0.0067 0.0123 MITT Number (%) 48.6% 18.4% 27.0% 20.5% 23.7% popu-of Patients lation Requiring Anti- Pseudo- monal Anti- microbials Hazard0.29 0.39 0.21 0.30 Ratio [0.12, [0.17, [0.09, [0.15, [95% CI] 0.71]0.87] 0.52] 0.60] P-value 0.0069 0.0215 0.0007 0.0007

The measured need for additional anti-pseudomonal microbials was reducedin all levofloxacin treatment groups. In addition, significant hazardratios were observed in all levofloxacin treatment groups. Hazard ratiosare related to the relative risk that an event may occur. The hazardratios were 0.29 for the levofloxacin 120 mg QD group, 0.39 for thelevofloxacin 240 mg QD group, and 0.21 for the levofloxacin 240 mg BIDgroup in the MITT Population compared to the placebo group, and werestatistically significant compared to placebo. FIGS. 5 and 6 are plotsof survival distribution function over time for each treatment group andshow that the survival distribution function for patients treated withplacebo begins to fall at a shorter time than patients treated withlevofloxacin. In sum, at least 240 mg BID showed significant efficacyover placebo at Day 28 in time to need for anti-pseudomonalantimicrobials.

Pulmonary Function Evaluations

Changes in FEV₁ (forced expiratory volume in 1 second), FVC (forcedvital capacity) and FEF 25-75 (forced expiratory flow 25-75%) from Day 1to all subsequent visits were determined for patients. Tables 26 and 27summarize the results for FEV₁ measurements in EE and MITT populations,respectively. FIGS. 7 and 8 show graphs of percent change in FEV₁ (L)and percent change in FEV₁(L) vs. placebo, respectively, at Day 28 forthe EET population treated with placebo, 120 mg QD, 240 mg QD, or 240 mgBID. FIG. 9 shows a graph of the categorical change in percent predictedFEV₁ at Day 28 for the EET population treated with 120 mg QD, 240 mg QD,or 240 mg BID. Tables 28 and 29 summarize the results for predicted FEVImeasurements in EE and MITT populations, respectively. Tables 30 and 31summarize results for FEF 25-75 measurements in EE and MITT populations,respectively.

TABLE 26 EE population—Changes in FEV₁values Com- bined Levo- Levo-Levo- Levo- floxacin Pla- floxacin floxacin floxacin 240 mg cebo 120 mg.240 mg 240 mg QD and (N = QD QD BID BID 32) (N = 35) (N = 35) (N = 34)(N = 69) Day 1 Mean 1.98 1.99 2.09 1.86 1.98 (SD) (0.61) (0.83) (0.75)(0.68) (0.72) Median 2.01 1.87 2.02 1.70 1.81 Day 14 Mean 1..97 2.002.16 2.02 2.09 (SD) (0.63) (0.83) (0.78) (0.76) (0.77) Median 1.93 1.822.09 1.88 2.03 Percent Mean −0.89 1.13 3.84 8.87 6.32 Change (SD) (6.86)(7.48) (10.23) (12.42) (11.56) from Base- Median −0.17 0.56 1.92 7.513.94 line P-value 0.4940 0.0737 0.001 0.0013 to Day vs. 14 Placebo Day28 Mean 1.95 2.03 2.13 1.90 2.02 (SD) (0.65) (0.89) (0.76) (0.68) (0.73)Median 1.91 1.91 2.15 11.79 1.98 Percent Mean −2.55 1.71 2.51 5.05 3.75Change (SD) (10.37) (9.37) (13.25) (11.49) (12.40) from Median −3.911.06 1.28 4.55 2.80 Base- P-value 0.1726 0.1121 0.0102 0.0168 line vs.to Day Placebo 28

TABLE 27 MITT population—Changes in FEV₁ values Com- bined Levo- Levo-Levo- Levo- floxacin Pla- floxacin floxacin floxacin 240 mg cebo 120 mg.240 mg 240 mg QD (N = QD QD BID and BID 37) (N = 38) (N = 37) (N = 39)(N =76) Day 1 Mean 1.94 1.95 2.05 1.88 1.96 (SD) (0.61) (0.81) (0.75)(0.68) (0.71) Median 1.97 1.83 1.90 1.70 1.80 Day 14 Mean 1.92 1.97 2.122.04 2.08 (SD) (0.63) (0.81) (0.78) (0.77) (0.77) Median 1.92 1.79 2.041.90 1.97 Percent Mean −1.21 1.87 3.81 9.34 6.61 Change (SD) (6.46)(9.41) (10.32) (13.72) (12.40) from Median −0.90 0.56 1.92 7.51 3.94Base- P-value 0.2873 0.0497 <.0001 0.0005 line to Day 14 Day 28 Mean1.89 1.99 2.09 11.93 2.01 (SD) (0.64) (0.88) (0.76) (0.69) (0.73) Median1.85 1.83 2.10 1.82 1.95 Percent Mean −2.96 1.92 2.35 5.93 4.14 Change(SD) (9.81) (9.95) (13.03) (15.07) (14.11) from Median −3.92 1.60 1.283.55 2.50 Base- P-value 0.1292 0.0831 0.0026 0.0063 line to Day 28

TABLE 28 EE population—Changes in % predicted FEV₁ Com- bined Levo-Levo- Levo- Levo- floxacin Pla- floxacin floxacin floxacin 240 mg cebo120 mg. 240 mg 240 mg QD (N = QD QD BID and BID 32) (N = 35) (N = 35) (N= 34) (N = 69) Day 1 Mean 54.4 54.0 55.9 48.2 52.1 (SD) (12.93) (17.79)(14.60) (14.88) (15.13) Median 54.5 53.0 56.0 45.5 51.0 Day 14 Mean 53.954.7 57.7 51.7 54.7 (SD) (13.21) (17.61) (15.19) (16.93) (16.23) Median54.5 53.0 62.0 49.5 52.0 Rel- Mean −0.77 1.77 3.57 7.67 5.59 ative (SD)(6.85) (6.08) (10.75) (14.70) (12.92) Percent Median 0.00 1.15 1.52 7.424.00 Change from Base- line to Day 14 Day 28 Mean 53.1 55.0 57.3 50.554.0 (SD) (14.05) (19.30) (15.52) (15.36) (15.71) Median 55.0 52.0 58.049.0 54.0 Rel- Mean −2.47 1.82 2.99 7.39 5.13 ative (SD) (10.34) (9.81)(14.54) (17.48) (16.06) Percent Median −3.74 2.30 0.00 4.88 4.00 ChangeP- 0.2655 0.1411 0.0034 0.0113 from value Base- line to Day 28

TABLE 29 MITT population—Changes in % predictedFEV₁ CombinedLevofloxacin Levofloxacin Levofloxacin Levofloxacin 240 mg 120 mg. 240mg 240 mg QD Placebo QD QD BID and BID (N = 37) (N = 38) (N = 37) (N =39) (N = 76) Day 1 Mean 52.4 52.9 55.4 48.8 52.0 (SD) (13.42 (17.68)(14.41) (15.15) (15.07) Median 53.0 53.0 56.0 46.0 51.0 Day 14 Mean 51.753.9 57.1 52.1 54.6 (SD) (13.84) (17.42) (14.96) (17.16) (16.20) Median53.0 53.0 61.0 49.55555 52.0 Mean −0.68 0.81 1.70 3.66 2.69 (SD) (3.37)(3.24) (5.39) (7.49) (6.57) Median −1.00 1.00 1.00 2.50 2.00 RelativeMean −1.30 2.46 3.51 8.30 5.93 Percent (SD) (6.58) (8.47) (10.83)(15.80) (13.70) Change Median −1.89 1.15 1.52 7.42 4.00 from P- 0.22840.0780 0.0005 0.0025 Baseline value to Day Day 28 Mean 50.9 54.1 56.750.8 53.8 (SD) (14.46) (19.19) (15.33) (15.40) (15.54) Median 52.0 50.054.0 49.0 53.5 Relative Mean −3.08 2.00 2.77 7.97 5.37 Percent (SD)(9.82) (10.07) (14.30) (19.49) (17.18) Change Median 3.77 2.30 0.00 4.443.85 from P- 0.1759 0.0888 0.0008 0.0036 Baseline value to Day 28

TABLE 30 EE population—Changes in FEF 25-75 values Com- bined Levo-Levo- Levo- Levo- floxacin Pla- floxacin floxacin floxacin 240 mg cebo120 mg. 240 mg 240 mg QD (N = QD QD BID and BID 32) (N = 35) (N = 35) (N= 34) (N = 69) Day 1 Mean 1.02 1.17 1.22 0.85 ( 1.04 (SD) (0.57) (0.78)(0.84) 0.53) (0.72 Median 0.94 0.98 1.10 0.68 0.83 Day 14 Mean 1.03 1.181.33 1.00 1.17 (SD) (0.64) (0.83) (0.93) (0.78) (0.87) Median 0.86 0.991.11 0.78 0.92 Percent Mean 1.95 0.41 9.58 16.99 13.23 Change (SD)(19.45) (12.28) (21.16) (32.60) (27.45) from Median −1.72 0.45 4.4111.66 8.79 Base- P- 0.6634 0.2532 0.0072 0.0275 line value to Day 14 Day28 Mean 1.00 1.23 1.27 0.93 1.10 (SD) (0.64) (0.93) (0.88) (0.59) (0.77)Median 0.94 1.03 1.15 0.78 0.97 Percent Mean −2.73 0.73 4.70 13.11 8.78Change (SD) (16.74) (15.76) (16.11) (28.50) (2:3.19) from Median −4.612.70 5.77 5.71 5.74 Base- P- 0.6038 0.2223 0.0007 0.0076 line value toDay 28

TABLE 31 MITT population−Changes in FEF 25-75 values Com- bined Levo-Levo- Levo- Levo- floxacin floxacin floxacin floxacin solution Pla-solution solution solution 240 mg cebo 120 mg. 240 mg 240 mg QD (N = QDQD BID and BID 37) (N = 38) (N=37) (N=39) (N = 76) Day 1 Mean 0.98 1.121.18 0.87 1.02 (SD) (0.55) (0.77) (0.83) (0.53) (0.71) Median 0.92 0.891.09 0.68 0.83 Day 14 Mean 0.97 1.15 1.29 1.01 1.15 (SD) (0.63) (0.82)(0.92) (0.78) (0.86) Median 0.81 0.85 1.08 0.78 0.90 Percent Mean −1.781.73 9.53 16.70 13.16 Change (SD) (22.86) (13.76) (20.70) (31.72)(26.92) from Median −4.46 1.35 4.41 11.66 8.79 Base- P- 0.5910 0.06990.0007 0.0027 line value to Day 14 Day 28 Mean 0.94 1.20 1.23 0.95 1.09(SD) (0.62) (0.92) (0.87) (0.61) (0.76) Median 0.91 0.83 1.12 0.82 0.93Percent Mean −6.40 1.94 5.02 14.19 9.611 Change (SD) (20.27) (16.55)(15.98) (28.56) (23.44) from Median −8.66 3.38 5.77 7.46 5.88 Base- P-0.1241 0.0489 <.0001 0.0003 line value to Day 28

In the EE population, FEV₁ values for patients administered levofloxacin240 mg BID increased from a median value of 1.70 L at Day 1 (baseline)to 1.79 L at Day 28, representing an increase in FEV₁ of approximately5%. In patients administered levofloxacin 240 mg QD, FEV₁ valuesincreased from a median value of 2.02 L on Day 1 to a median value of2.15 L on Day 28, representing an increase in FEV₁ of approximately 6%.In patients administered levofloxacin 120 mg QD, FEV₁ values increasedfrom a median value of 1.87 L on Day 1 to a median value of 1.91 L onDay 28, representing an increase in FEV₁ of approximately 2%. Thelargest differences for FEV₁ between patients administered placebo andpatients administered levofloxacin were 0.16 L on Day 14, and 0.24 L onDay 28.

In the MITT population, FEV₁ values for patients administeredlevofloxacin 240 mg BID increased from a median value of 1.70 L at Day 1(baseline) to 1.82 L at Day 28, representing an increase in FEV₁ ofapproximately 7%. In patients administered levofloxacin 240 mg QD, FEV₁values increased from a median value of 1.90 L on Day 1 to a medianvalue of 2.10 L on Day 28, representing an increase in FEVI ofapproximately 10%. The largest differences for FEV₁ between patientsadministered placebo and patients administered levofloxacin were 0.12 Lon Day 14, and 0.25 L on Day 28.

FEF 25-75 values relate to the average flow of air coming out of thelungs during the middle portion of the expiration. In small airwaydiseases this value can be reduced. In the EE population, FEF 25-75values for patients administered levofloxacin 240 mg BID increased froma median value of 0.68 at Day 1 (baseline) to 0.78 at Day 28,representing an increase in FEF 25-75 of approximately 15%. In patientsadministered levofloxacin 240 mg QD, FEF 25-75 values increased from amedian value of 1.10 on Day 1 to a median value of 1.15 on Day 28,representing an increase in FEF 25-75 of approximately 4%. In patientsadministered levofloxacin 120 mg QD, FEF 25-75 values increased from amedian value of 0.98 on Day 1 to a median value of 1.03 on Day 28,representing an increase in FEF 25-75 of approximately 5%. The largestdifferences for FEF 25-75 between patients administered placebo andpatients administered levofloxacin were 0.25 L on Day 14, and 0.21 L onDay 28.

In the MITT population, FEF 25-75 values for patients administeredlevofloxacin 240 mg BID increased from a median value of 0.68 at Day 1(baseline) to 0.82 at Day 28, representing an increase in FEF 25-75 ofapproximately 20%. In patients administered levofloxacin 240 mg QD, FEF25-75 values increased from a median value of 1.09 on Day 1 to a medianvalue of 1.12 on Day 28, representing an increase in FEF 25-75 ofapproximately 3%. The largest differences for FEF 25-75 between patientsadministered placebo and patients administered levofloxacin were 0.27 Lon Day 14, and 0.21 L on Day 28.

Safety Evaluations

Adverse events and drug intolerability from Day 1 through the end ofstudy were evaluated. No significant adverse events were reported.

Flouroquinolone-induced arthralgia and myalgia have been previouslyreported in the use of some fluoroquinolones, for example in thetreatment of sinusitis (0-Lee T., et al “Fluoroquinolone-inducedarthralgia and myalgia in the treatment of sinusitis” Am. J. Rhinol.(2005) 19:395-9, incorporated by reference in its entirety). In thisstudy, Arthralgia was reported in 5.4% of patients administered withplacebo. No arthralgia was reported in patients administered anylevofloxacin formulations.

Example 3-Phase I Clinical Study in Pediatric CF Patients

A Phase 1 multicenter, open-label study was carried out to evaluate thesafety, tolerability and pharmacokinetics of weight-adjusted doses oflevofloxacin formulated with MgCl₂ administered once daily for 14 daysto stable pediatric CF patients. Patients were divided into 2 groupsbased on their age: 6-11 years of age and 12-16 years of age. The dailydose administered of levofloxacin formulated with MgCl₂ was divided asfollows: patients that weighed 14-21 kg received a 120 mg dose, patientsthat weighed 22-30 kg received a 180 mg dose, and patients that weighedmore than 30 kg received a 240 mg dose. A total of 27 patients wereenrolled and all patients completed the study. There were 14 patients inthe 6-11 years of age group and 13 patients in the 12-16 years of agegroup. Seven patients (all in the 6-11 age group) received 180 mg QDlevofloxacin formulated with MgCl₂ and the remaining 20 patientsreceived 240 mg QD levofloxacin formulated with MgCl₂. FIG. 10 shows agraph of dose normalized serum AUC in pediatric CF patients vs. patientbody weight. FIGS. 11A-9B show graphs of dose normalized serum AUC inpediatric CF patients vs. patient age, and vs. BSA, respectively. FIGS.11C-9E show graphs of dose normalized serum Cmax in pediatric CFpatients vs. patient body weight, vs. patient age, and vs. BSA,respectively.

Serum levofloxacin exposures with either the 180 or 240 mg dose oflevofloxacin formulated with MgCl₂ appear to be in the ranges observedin adults CF patients studied in a related clinical trial (data notshown).

Example 4-Phase III Clinical Study

A Phase 3, multi-center, multinational, randomized, double-blind,placebo-controlled study to evaluate the efficacy and safety oflevofloxacin formulated with MgCl₂ in stable CF patients is performed.Following a 14-day screening period, patients are randomized at Visit1/Day 1 in a 2:1 ratio to receive levofloxacin formulated with MgCl₂ orplacebo. Randomization is stratified by geographic region (US vs.non-US), age (12-18 years vs. >18 years of age) and by FEV₁ percentpredicted (<55% vs. >55%). Patients receive 28 days of eitherlevofloxacin formulated with MgCl₂ or placebo followed by 28 days ofobservation. Patients should remain off anti-pseudomonal antimicrobials,other than Study Drug (levofloxacin formulated with MgCl₂ or placebo)and maintenance oral azithromycin (if applicable), for the duration ofthe study unless they meet the protocol defined definition of anexacerbation or unless determined to be necessary for safety reasons bythe Investigators. The end of the study is defined as the last visit ofthe last patient.

Levofloxacin formulated with MgCl₂ (levofloxacin inhalation solution,Aeroquin™) will be provided in single use ampules ready foradministration. Each ampule contains 240 mg of levofloxacin formulatedwith MgCl₂ in 2.4 mL (100 mg/mL). A 240 mg dose (1 ampule) isadministered using a PART investigational eFlow® nebulizer twice daily(BID) approximately 8-12 hours apart. Placebo is riboflavin 5′-phosphate(solubilized form of vitamin B2) in 0.9% saline provided in single useampules ready for administration. Each ampule contains 9.6 lag ofriboflavin 5′-phosphate in a volume of 2.4 mL. A PART investigationaleFlow® nebulizer is optimized and customized for use only withlevofloxacin formulated with MgCl₂. Both levofloxacin formulated withMgCl₂ and the placebo control are administered using this nebulizersystem. Analysis populations include: (1) safety/modified intent totreat (MITT) population (all patients enrolled in the study who receiveat least one dose of Study Drug (levofloxacin formulated with MgCl₂ orplacebo); (2) efficacy evaluable Population (all patients enrolled inthe study, without major protocol violations, who receive at least 80%of Study Drug (levofloxacin formulated with MgCl₂/placebo) doses); and(3) pharmacokinetic population (all patients who receive a least onedose of Study Drug (levofloxacin formulated with MgCl₂/placebo) and haveat least one pharmacokinetic (PK) blood or sputum sample collected).

Inclusion criteria for study patients included: at least 12 years ofage; weigh at least 30 kg or 66 pounds; and have documented CF diagnosisas evidenced by one or more clinical features consistent with the CFphenotype. Exclusion criteria for study patients included: use of aninvestigational agent within 28 days prior to Visit 1; use of anynebulized or systemic antimicrobials active against P. aeruginosa within28 days prior to Visit 1, other than maintenance oral azithromycin,which must have been initiated at least 28 days prior to Visit 1; anduse of oral corticosteroids in doses exceeding the equivalent of 10 mgprednisone/day or 20 mg prednisone every other day at Screening or Visit1.

Primary Efficacy Evaluations

The primary endpoint includes the time (in days) to an exacerbation fromBaseline (Visit 1/Day 1) until Final Visit. To meet the endpoint apatient must concurrently meet 4 of the 12 symptoms/signs that make upthe Fuchs definition of an exacerbation (Fuchs H J, et al. Effect ofaerosolized recombinant human DNase on exacerbations of respiratorysymptoms and on pulmonary function in patients with cystic fibrosis. NEngl J Med 1994; 331:637-642, incorporated by reference in itsentirety).

The 12 symptoms/signs defined by the Fuchs criteria include: change insputum; new or increased hemoptysis; increased cough; increased dyspnea;malaise, fatigue or lethargy; temperature above 38° C.; anorexia orweight loss; sinus pain or tenderness; change in sinus discharge; changein physical examination of the chest; decrease in pulmonary function by10% or more from a previously recorded value; and radiographic changesindicative of pulmonary infection. The above symptoms associated with anexacerbation are recorded from each patient using a standardizedquestionnaire, the Respiratory and Systemic Symptoms Questionnaire(RSSQ) at patient visits. Changes the patient experiences are relativeto what the patient considers normal on a day-to-day basis. It isanticipated that patients administered aerosolized levofloxacinformulated with MgCl₂ provide the following signs/symptoms that areincluded in the Fuchs criteria and that are recorded using the RSSQ: (1)Increased sputum production: patients have no change, a little less ormuch less amounts of sputum when coughing up. (2) Change in sputumappearance: for sputum thickness, patients have a little thinner or muchthinner sputum; for sputum color, patients have a better color of sputum(better increases from brown->green->yellow->clear). (3) Increased chestcongestion: patients have a little decrease, or a large decrease inchest congestion. (4) New or increased coughing up of blood: patientshave a little decrease or a large decrease in the amount of bloodcoughed up. (5) Increased cough: for intensity of cough, patients have alittle lighter, or much lighter coughs; for frequency of cough, patientscough a little less often or much less often. (6) Decreased exercisetolerance: patients perform daily activities, e.g., climbing stairs, alittle easier, or much easier. (7) Increased dyspnea with exertion:patients breathe a little easier or much easier when performing dailyactivities. (8) Malaise, fatigue or lethargy: patients have a littlemore energy or much more energy since last visit. (9) Fever: patientshave no fever since last visit. (10) Weight loss: patients have nochange in weight, or a little weight gain. (11) Sinus pain andtenderness: patient has no pain or tenderness. (12) Change in sinusdischarge: patient has better sinus discharge (a decrease in thicknessand/or better color). (13) School or work absenteeism (due to illness):patient has no absenteeism from schedules activities. (14) Decreasedappetite: patient has a little increase in appetite.

Secondary Efficacy Evaluations

Secondary Endpoints include clinical, pulmonary function, microbiology,and patient reported outcome characteristics.

Clinical Characteristics

Clinical characteristics include the time (in days) to administration ofsystemic (oral or IV) and/or inhaled anti-pseudomonal antimicrobialsfrom Baseline (Visit 1/Day 1) until Final Visit. To meet this endpoint,patients must have at least one of four worsening respiratory symptoms(increased cough, increased sputum/chest congestion, decreased exercisetolerance, decreased appetite) at the time of administration of theanti-pseudomonal antimicrobial agent. Clinical characteristics alsoinclude the proportion of patients who miss at least 1 day ofschool/work secondary to worsening respiratory status.

It is anticipated that patients administered aerosolized levofloxacinformulated with MgCl₂ have an increased time to administration ofsystemic (oral or IV) and/or inhaled anti-pseudomonal antimicrobialsfrom Baseline (Visit 1/Day 1) until Final Visit compared to patientsadministered placebo. In addition, the proportion of patientsadministered aerosolized levofloxacin formulated with MgCl₂ who miss atleast 1 day of school/work secondary to worsening respiratory status isless than the proportion of patients administered placebo who miss atleast 1 day of school/work secondary to worsening respiratory status.

Pulmonary Function Characteristics

Pulmonary function characteristics include: percent change in FEV₁ (L)from Baseline to Day 28; relative change in FEV₁ (percent predicted)from Baseline to Day 28; percent change in FEF25-75 (L/s) from Baselineto Day 28; percent change in FVC (L) from Baseline to Day 28; andcategorical assessment of percent change in FEV₁(L) and relative changein percent predicted FEV₁ from Baseline to Day 28.

It is anticipated that patients administered aerosolized levofloxacinformulated with MgCl₂ have more advantageous pulmonary functioncharacteristics compared to patients administered placebo.

Microbiology Characteristics

Microbiology characteristics include: change in P. aeruginosa density(log 10 colony-forming units [CFU] per gram sputum) from Baseline to Day28; categorical assessment of change in P. aeruginosa density (log 10colony-forming units [CFU] per gram sputum) from Baseline to Day 28; andchange in Stenotrophomonas sp., Achromobacter sp., Burkholderia sP. andS. aureus density (log 10 colony-forming units [CFU] per gram sputum)from Baseline to Day 28.

It is anticipated that patients administered aerosolized levofloxacinformulated with MgCl₂ have more advantageous microbiologycharacteristics, e.g., decreased P. aeruginosa sputum density, decreasedStenotrophomonas sp., Achromobacter sp., Burkholderia sP. and S. aureussputum density, compared to patients administered placebo.

Patient Reported Outcome Characteristics

Patient reported outcome characteristics include: change in therespiratory domain of the CFQ-R from Baseline to Day 28; and categoricalassessment of change in the respiratory domain of the CFQ-R fromBaseline to Day 28.

It is anticipated that patients administered aerosolized levofloxacinformulated with MgCl₂ have more advantageous patient reported outcomecharacteristics than patients administered placebo.

Example 5—In Vivo Antibacterial Activity of Levofloxacin InhalationSolution Against Burkholderia cepacia

Burkholderia cepacia is an opportunistic pathogen capable of causingpulmonary infection in CF patients. Infections with certain strains ofB. cepacia cause “cepacia syndrome” which is characterized byprogressive and invasive necrotizing pneumonia and septicemia. Most B.cepacia have high MICs to many antibiotics. Aerosolized levofloxacinformulated with MgCl₂ enables aerosol delivery of high drugconcentrations to the lung.

Five B. cepacia strains with levofloxacin MICs ranging between 0.25-8mg/L were tested in a mouse model of pulmonary infection. Female BALB/cmice were injected with 150 mg/kg of cyclophosphamide 3 days prior toinfection. On day 4, the mice were infected with 50 ul of bacterialsuspension (˜10⁶ CFU/ml) using a curved bead-tipped oral gavage syringeunder isoflurane anesthesia. Treatment with levofloxacin formulated withMgCl₂ (60 mg/kg BID) or saline only was initiated 72 hourspost-infection and continued twice daily for four days using amicrospray aerosol device. Sixteen hours after the last treatment, micewere sacrificed, lungs harvested, homogenized, and plated to determinecolony counts (CFU).

As part of a Phase 2b trial of aerosolized levofloxacin formulated withMgCl₂ in CF patients, a 16 year old male patient infected with B.cepacia (levofloxacin MIC=>128 mg/L) received levofloxacin formulatedwith MgCl₂ 240 mg QD for 28 days.

Aerosolized levofloxacin formulated with MgCl₂ produced at least one logCFU of bacterial killing for all strains in the mouse infection model(Table 32). In the CF patient, a 1.7 log CFU decrease in bacterialcounts was observed over 28 days.

TABLE 32 Genomovar II Genomovar III Strains BC1012 BC1013 BC1014 BC1020BC1021 MIC (mg/L) 4 0.25 1 8 8 Mean Change in −1.03 −2.08 −1.47 −1.11−1.35 Log CFU/Lungs

An increase in the CF patient's FEV₁ was observed. On day 1 of thestudy, the patient's FEV₁ was 1.21 L, this increased to 1.30 L on day28, a 7% improvement. From a population model, Cmax and AUC values werecalculated to be approximately 12,900 mg/L and 4,400 mg*h/L,respectively.

Aerosol administration of levofloxacin formulated with MgCl₂ producedsignificant bacterial killing in strains with a wide range of MICs. Thenon-clinical and clinical data support the future clinical evaluation oflevofloxacin formulated with MgCl₂ in the management of chronicpulmonary infections due to B. cepacia.

To the extent publications and patents or patent applicationsincorporated by reference herein contradict the disclosure contained inthe specification, the specification is intended to supersede and/ortake precedence over any such contradictory material.

Unless otherwise defined, all terms (including technical and scientificterms) are to be given their ordinary and customary meaning to a personof ordinary skill in the art, and are not to be limited to a special orcustomized meaning unless expressly so defined herein.

Terms and phrases used in this application, and variations thereof,unless otherwise expressly stated, should be construed as open ended asopposed to limiting. As examples of the foregoing, the term ‘including’should be read to mean ‘including, without limitation’ or the like; theterm ‘comprising’ as used herein is synonymous with ‘including,’containing,’ or ‘characterized by,’ and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps: theterm ‘example’ is used to provide exemplary instances of the item indiscussion, not an exhaustive or limiting list thereof; adjectives suchas ‘known’, ‘normal’, ‘standard’, and terms of similar meaning shouldnot be construed as limiting the item described to a given time periodor to an item available as of a given time, but instead should be readto encompass known, normal, or standard technologies that may beavailable or known now or at any time in the future; and use of termslike ‘preferably,’ preferred,” desired,’ or ‘desirable,’ and words ofsimilar meaning should not be understood as implying that certainfeatures are critical, essential, or even important to the structure orfunction of the invention, but instead as merely intended to highlightalternative or additional features that may or may not be utilized in aparticular embodiment of the invention. Likewise, a group of itemslinked with the conjunction ‘and’ should not be read as requiring thateach and every one of those items be present in the grouping, but rathershould be read as ‘and/or’ unless expressly stated otherwise. Similarly,a group of items linked with the conjunction ‘or’ should not be read asrequiring mutual exclusivity among that group, but rather should be readas ‘and/or’ unless expressly stated otherwise. In addition, as used inthis application, the articles ‘a’ and ‘an’ should be construed asreferring to one or more than one (i.e., to at least one) of thegrammatical objects of the article. By way of example, ‘an element’means one element or more than one element.

The presence in some instances of broadening words and phrases such as‘one or more’, ‘at least’, ‘but not limited to’, or other like phrasesshall not be read to mean that the narrower case is intended or requiredin instances where such broadening phrases may be absent.

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification are to be understood as beingmodified in all instances by the term ‘about.’ Accordingly, unlessindicated to the contrary, the numerical parameters set forth herein areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of anyclaims in any application claiming priority to the present application,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Furthermore, although the foregoing has been described in some detail byway of illustrations and examples for purposes of clarity andunderstanding, it is apparent to those skilled in the art that certainchanges and modifications may be practiced. Therefore, the descriptionand examples should not be construed as limiting the scope of theinvention to the specific embodiments and examples described herein, butrather to also cover all modification and alternatives coming with thetrue scope and spirit of the invention.

1.-20. (canceled)
 21. A method for treating a pulmonary infection,wherein the pulmonary infection comprises one or more Mycobacterium, themethod comprising: administering via inhalation 240 mg of levofloxacintwice daily for 28 days to the human having cystic fibrosis to treat theMycobacterium pulmonary infection; wherein the levofloxacin is in anaerosol of a solution comprising levofloxacin at a concentration fromabout 90 mg/ml to about 110 mg/ml, a magnesium cation at a concentrationfrom about 175 mM to about 225 mM, wherein the solution has a pH fromabout 5 to about 7, and an osmolality from about 300 mOsmol/kg to about500 mOsmol/kg.
 22. The method of claim 21, wherein the Mycobacterium isMycobacterium tuberculosis, Mycobacterium avium, Mycobacteriumintracellulare, and Mycobacterium leprae, Chlamydia pneumoniae,Mycoplasma pneumoniae
 23. The method of claim 22, wherein theMycobacterium is Mycobacterium avium, Mycobacterium intracellulare, or acombination thereof.
 24. The method of claim 21, wherein the magnesiumcation is in the form of magnesium chloride.
 25. The method of claim 21,wherein the solution comprises a levofloxacin concentration of about 100mg/ml, a magnesium chloride concentration of about 200 mM, a pH betweenabout 6.0 to about 6.5, and an osmolality of between about 300 mOsmol/kgto about 500 mOsmol/kg, and lacks lactose.
 26. The method of claim 21,wherein the aerosol of the solution comprises a mass median aerodynamicdiameter from about 2 microns to about 5 microns with a geometricstandard deviation less than or equal to 2.5 microns.
 27. The method ofclaim 21, wherein the aerosol is produced by a vibrating mesh nebulizer.28. The method of claim 21, further comprising administering anantibiotic, a bronchodilator, an anticholinergic agent, aglucocorticoid, an eicosanoid inhibitor, or a combination of two or morethereof.